Methods for bead-based sequencing

ABSTRACT

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/628,209 filed Nov. 30, 2009, which is a continuation of U.S.application Ser. No. 12/220,201 filed Jul. 21, 2008, which is acontinuation of U.S. application Ser. No. 11/345,979 filed Feb. 1, 2006,and claims priority to U.S. application No. 60/722,526 filed Sep. 30,2005, U.S. application No. 60/699,541 filed Jul. 15, 2005, U.S.application No. 60/673,749 filed Apr. 21, 2005, U.S. application No.60/656,599 filed Feb. 25, 2005, and U.S. application No. 60/649,294filed Feb. 1, 2005, all of which disclosures are herein incorporated byreference in their entirety.

REFERENCE TO BIOLOGICAL SEQUENCE DISCLOSURE

This application contains nucleotide sequence and/or amino acid sequencedisclosure in computer readable form and a written sequence listing, theentire contents of both of which are expressly incorporated by referencein their entirety as though fully set forth herein.

BACKGROUND OF THE INVENTION

Nucleic acid sequencing techniques are of major importance in a widevariety of fields ranging from basic research to clinical diagnosis. Theresults available from such technologies can include information ofvarying degrees of specificity. For example, useful information canconsist of determining whether a particular polynucleotide differs insequence from a reference polynucleotide, confirming the presence of aparticular polynucleotide sequence in a sample, determining partialsequence information such as the identity of one or more nucleotideswithin a polynucleotide, determining the identity and order ofnucleotides within a polynucleotide, etc.

DNA strands are typically polymers composed of four types of subunits,namely deoxyribonucleotides containing the bases adenine (A), cytosine(C), guanine (G), and thymidine (T). These subunits are attached to oneanother by covalent phosphodiester bonds that link the 5′ carbon of onedeoxyribose group to the 3′ carbon of the following group. Mostnaturally occurring DNA consists of two such strands, which are alignedin an antiparallel orientation and are held together by hydrogen bondsformed between complementary bases, i.e., between A and T and between Gand C.

DNA sequencing first became possible on a large scale with thedevelopment of the chain termination or dideoxynucleotide method(Sanger, et al., Proc. Natl. Acad. Sci. 74:5463-5467, 1977) and thechemical degradation method (Maxam & Gilbert, Proc. Natl. Acad. Sci.74:560-564, 1977), of which the former has been most extensivelyemployed, improved upon, and automated. In particular, the use offluorescently labeled chain terminators was of key importance in thedevelopment of automatic DNA sequencers. Common to both of the aboveapproaches is the production of one or more collections of labeled DNAfragments of differing sizes, which must then be separated on the basisof length to determine the identity of the nucleotide at the 3′ end ofthe fragment (in the chain termination method) or the identity of thenucleotide that was most recently removed from the fragment (in the caseof the chemical degradation method).

Although currently available sequencing technologies have allowed theachievement of major landmarks such as the sequencing of a number ofcomplete genomes, these techniques have a number of disadvantages, andconsiderable need for improvement remains in a number of areas.Separation of labeled DNA fragments has typically been achieved usingpolyacrylamide gel electrophoresis. However, this step has proven to bea major bottleneck limiting both the speed and accuracy of sequencing inmany contexts. While capillary electrophoresis (CAE) proved to be thebreakthrough that allowed the completion of the Human Genome Project(Venter, et al., Science, 291:1304-1351, 2001; Lander, et al., Nature,409:860-921, 2001), significant shortcomings remain. For example, CAEstill requires a time-consuming separation step and still involvesdiscrimination based on size, which can be inaccurate.

A variety of alternatives to the chain termination method have beenproposed. In one approach, often referred to as “sequencing bysynthesis”, an oligonucleotide primer is first hybridized to a targettemplate. The primer is then extended by successive cycles ofpolymerase-catalyzed addition of differently labeled nucleotides, whoseincorporation into the growing strand is detected. The identity of thelabel serves to identify the complementary nucleotide in the template.Alternately, multiple reactions can be performed in parallel using eachof the nucleotides, and incorporation of a labeled nucleotide in thereaction that uses a particular nucleotide identifies the complementarynucleotide in the template. (See, e.g., Melamede, U.S. Pat. No.4,863,849; Cheeseman, U.S. Pat. No. 5,302,509, Tsien et al,International application WO 91/06678; Rosenthal et al, Internationalapplication WO 93/21340; Canard et al, Gene, 148: 1-6 (1994); Metzker etal, Nucleic Acids Research, 22: 4259-4267 (1994)).

To efficiently sequence polynucleotides of any significant length, it isdesirable that the polymerase incorporates exactly one nucleotide ineach cycle. Therefore it is generally necessary to use nucleotides thatact as chain terminators, i.e., their incorporation prevents furtherextension by the polymerase. The incorporated nucleotide must then bemodified, either enzymatically or chemically, to allow the polymerase toincorporate the next nucleotide. A variety of nucleotide analogs thatcan serve as chain terminators but can be modified after theirincorporation such that they can be extended in a subsequent step havebeen proposed. Such “reversible terminators” have been described, forexample, in U.S. Pat. Nos. 5,302,509; 6,255,475; 6,309, 836;6,613,513.However, it has proven difficult to identify reversible terminators thatcan be incorporated by polymerase with high efficiency, probably due tothe fact that given the small size of a nucleotide, modifications thataffect the ability of the nucleotide to act as a terminator also affectits incorporation into a growing polynucleotide strand.

Other sequencing approaches include pyrosequencing, which is based onthe detection of the pyrophosphate (PPi) that is released during DNApolymerization (see, e.g., U.S. Pat. Nos. 6,210,891 and 6,258,568. Whileavoiding the need for electrophoretic separation, pyrosequencing suffersfrom a large number of drawbacks that have as yet limited its widespreadapplicability (França, et al., Quarterly Reviews of Biophysics,35(2):169-200, 2002). Sequencing by hybridization has also been proposedas an alternative (U.S. Pat. No. 5,202,231; WO 99/60170; WO 00/56937;Drmanac, et al., Advances in Biochemical Engineering/Biotechnology,77:76-101, 2002) but has a number of disadvantages including thepotential for error in discriminating between highly similar sequences.Single-molecule sequencing by exonuclease, which involves labeling everybase in one strand and then detecting sequentially cleaved 3′ terminalnucleotides in a sample stream is theoretically a very powerful methodfor rapidly determining the sequence of a long DNA molecule (Stephan, etal., J. Biotechnol., 86:255-267, 2001). However, various technicalhurdles remain to be overcome before realization of this potential(Stephan, et al., 2001).

Diagnostic tests based upon particular sequence variations are alreadyin use for a variety of different diseases. The sequencing of the humangenome is widely thought to herald an era of personalized medicine inwhich therapies, including preventive therapies, will be tailored to theparticular genetic make-up of the patient or will be selected based uponthe identification of particular alleles or mutations. There is anincreasing need for rapid and accurate determination of sequencevariants of pathogenic agents such as HIV. Thus it is evident that thedemand for accurate and rapid sequence determination will expand greatlyin the immediate future. Improved methods for sequence determination ofall types are therefore needed.

SUMMARY OF THE INVENTION

The present invention provides new and improved sequencing methods thatavoid the necessity for performing fragment separation and also incertain embodiments avoid the need to use polymerase enzymes. Analternative to the methods discussed in the Background is described inU.S. Pat. Nos. 5,750,341 and 6,306,597, to Macevicz. The methods arebased on repeated cycles of duplex extension along a single-strandedtemplate. In preferred embodiments of these methods a nucleotide isidentified in each cycle. The present invention provides improvements tothese methods. The improvements allow efficient implementation of themethods and are particularly suited for high throughput sequencing. Inaddition, the invention provides methods for sequence determination thatinvolve repeated cycles of duplex extension along a single-strandedtemplate but do not involve identification of any individual nucleotideduring each cycle.

In one aspect, the invention provides improved methods for sequencingbased on successive cycles of duplex extension along a single-strandedtemplate, ligation of labeled extension probes, and detection of thelabel. In general, extension starts from a duplex formed by aninitializing oligonucleotide and a template. The initializingoligonucleotide is extended by ligating an oligonucleotide probe to itsend to form an extended duplex, which is then repeatedly extended bysuccessive cycles of ligation. During each cycle, the identity of one ormore nucleotides in the template is determined by identifying a label onor associated with a successfully ligated oligonucleotide probe. Thelabel of the newly added probe can also be detected prior to ligation,instead of, or in addition to, after ligation. Generally it is preferredto detect the label after ligation.

In preferred embodiments the probe has a non-extendable moiety in aterminal position (at the opposite end of the probe from the nucleotidethat is ligated to the growing nucleic acid strand of the duplex) sothat only a single extension of the extended duplex takes place in asingle cycle. By “non-extendable” is meant that the moiety does notserve as a substrate for ligase without modification. For example, themoiety may be a nucleotide residue that lacks a 5′ phosphate or 3′hydroxyl group. The moiety may be a nucleotide with a blocking groupattached thereto that prevents ligation. In preferred embodiments of theinvention the non-extendable moiety is removed after ligation toregenerate an extendable terminus so that the duplex can be furtherextended in subsequent cycles.

To allow removal of the non-extendable moiety, in certain embodiments ofthe invention the probe contains at least one internucleoside linkagethat can be cleaved under conditions that will not substantially cleavephosphodiester bonds. Such linkages are referred to herein as “scissileinternucleosidic linkages” or “scissile linkages”. Cleavage of thescissile internucleosidic linkage removes the non-extendable moiety andeither regenerates an extendable probe terminus or leaves a terminalresidue that can be modified to form an extendable probe terminus. Thescissile internucleosidic linkage may be located between any twonucleosides in the probe. Preferably the scissile linkage is located atleast several nucleotides away from (i.e., distal to) the newly formedbond. The nucleotides in the extension probe between the terminalnucleotide that is ligated to the extendable terminus and the scissilelinkage need not hybridize perfectly to the template. These nucleotidesmay serve as a “spacer” and allow identification of nucleotides locatedat intervals along the template without performing a cycle for eachnucleotide within the interval.

The scissile internucleosidic linkage and the label are preferablylocated such that cleavage of the scissile internucleosidic linkageseparates the extension probe into a labeled portion and a portion thatremains part of the growing nucleic acid strand, allowing the labeledportion to diffuse away (e.g., upon raising the temperature). Forexample, the label may be attached to the terminal nucleotide of theextension probe, at the opposite end from the nucleotide that isligated. Alternately, the label may be removed using any of a number ofapproaches.

The present inventors have discovered that phosphorothiolate linkages,in which one of the bridging oxygen atoms in the phosphodiester bond isreplaced by a sulfur atom, are particularly advantageous scissileinternucleosidic linkages. The sulfur atom in the phosphorothiolatelinkage may be attached to either the 3′ carbon of one nucleoside or the5′ carbon of the adjacent nucleoside.

In certain embodiments of the methods described above a plurality ofsequencing reactions is performed. The reactions use initializingoligonucleotides that hybridize to different sequences of the templatesuch that the terminus at which the first ligation occurs is located atdifferent positions with respect to the template. For example, thelocations at which the first ligation occurs may be shifted, or “out ofphase”, relative to one another by 1 nucleotide increments. Thus aftereach cycle of extension with oligonucleotide probes of the same length,the same relative phase exists between the ends of the initializingoligonucleotides on the different templates. The reactions can beperformed in parallel, in separate compartments each containing copiesof the same template, or in series, i.e., by removing the extendedduplex from the template after obtaining sequence information using afirst initializing oligonucleotide and then performing additionalreaction(s) using initializing oligonucleotides that hybridize todifferent sequences of the template.

In another aspect, the invention provides solutions that are of use fora variety of nucleic acid manipulations. In one embodiment, theinvention provides a solution containing or consisting essentially of1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium bisulfate (NaHSO₄) inwater. The solution may contain or consist essentially of about 2% SDS,about 200 mM NaCl, and about 10 mM sodium bisulfate (NaHSO₄) in water.For example, in one embodiment the solution contains 2% SDS, 200 mMNaCl, and 10 mM sodium bisulfate (NaHSO₄) in water. In anotherembodiment the solution consists essentially of 2% SDS, 200 mM NaCl, and10 mM sodium bisulfate (NaHSO₄) in water. In certain embodiments thesolution has a pH between 2.0 and 3.0, e.g., 2.5. The solutions areuseful to separate double-stranded nucleic acids, e.g., double-strandedDNA, into individual strands, i.e., to denature (melt) double-strandednucleic acids. In certain embodiments both strands are DNA. In otherembodiments both strands are RNA. In other embodiments one strand is DNAand the other strand is RNA. In other embodiments one or both strandscontains both RNA and DNA. In other embodiments one or both of thestrands contains at least one nucleotide other than A, G, C, or T. Insome embodiments one or both of the strands contains a non-naturallyoccurring nucleotide. In yet other embodiments one or more of theresidues is a trigger residue, e.g., an abasic residue or damaged base.In some embodiments one or more residues contains a universal base. Insome embodiments one or both of the strands contains a scissile linkage.

The double-stranded nucleic acids may be fully or partiallydouble-stranded. They may be free in solution or one or both strands maybe physically associated with (e.g., covalently or noncovalentlyattached to) a solid or semi-solid support or substrate. Of particularnote, double-stranded nucleic acids incubated in these solutions areeffectively separated into single strands in the absence of heat orharsh denaturants that could cause gel delamination (e.g., when thenucleic acids are located in or attached to a semi-solid support such asa polyacrylamide gel) or could disrupt noncovalent associations such asstreptavidin (SA)-biotin association (e.g., when the nucleic acids areattached to a support or substrate via a SA-biotin association). In oneembodiment the solutions are used to separate double-stranded nucleicacids wherein one of the nucleic acids is attached to a bead via aSA-biotin association.

The invention also provides a method of separating strands of adouble-stranded nucleic acid comprising the step of: contacting thedouble stranded nucleic acid with any of the afore-mentioned solutions,e.g., an aqueous solution containing about 1.0-3.0% SDS, about 100-300mM NaCl, and about 5-15 mM sodium bisulfate (NaHSO₄), e.g., containing1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium bisulfate (NaHSO₄). Inone embodiment the solution contains about 2% SDS, 200 mM NaCl, and 10mM sodium bisulfate (NaHSO₄), e.g., 2% SDS, 200 mM NaCl, and 10 mMsodium bisulfate (NaHSO₄). In another embodiment the solution consistsessentially of 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO₄)in water. In certain embodiments the solution has a pH between 2.0 and3.0, e.g., 2.5. In some embodiments the double-stranded nucleic acid isincubated in the solution. In other embodiments the double-strandednucleic acid (preferably attached to a support or substrate) is washedwith the solution. In some embodiments the double-stranded nucleic acidis contacted with the solution for a time sufficient to separate atleast 10% of the double-stranded nucleic acid molecules into singlestrands. In some embodiments the double-stranded nucleic acid iscontacted with the solution for a time sufficient to separate at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of thedouble-stranded nucleic acids into single strands. In an exemplaryembodiment the double-stranded nucleic acid is contacted with thesolution for between 15 seconds and 3 hours. In another embodiment thedouble-stranded nucleic acid is contacted with the solution for between1 minute and 1 hour. In certain embodiments the double-stranded nucleicacid is contacted with the solution for about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or 60 minutes. The methods may comprise afurther step of removing the solution or removing some or all of thenucleic acids from the solution following a period of incubation.

The solutions find use in one or more steps of a number of thesequencing methods described herein and may be employed in any of thesemethods. For example, the solutions may be used to separate an extendedduplex from a template. The solutions may be used following cleavage ofa scissile linkage to remove the portion of an extension probe that isno longer attached to the extended duplex. The solutions are also of usein separating strands of a triple-stranded nucleic acids or inseparating double-stranded regions of a single nucleic acid strand thatcontains self-complementary portions that have hybridized to oneanother.

In another aspect, the invention provides methods for obtaininginformation about a sequence using a collection of at least twodistinguishably labeled oligonucleotide probe families. The probes inthe probe families contain an unconstrained portion and a constrainedportion. As in the methods described above, extension starts from aduplex formed by an initializing oligonucleotide and a template. Theinitializing oligonucleotide is extended by ligating an oligonucleotideprobe to its end to form an extended duplex, which is then repeatedlyextended by successive cycles of ligation. The probe has anon-extendable moiety in a terminal position (at the opposite end of theprobe from the nucleotide that is ligated to the growing nucleic acidstrand of the duplex) so that only a single extension of the extendedduplex takes place in a single cycle. During each cycle, a label on orassociated with a successfully ligated probe is detected, and thenon-extendable moiety is removed or modified to generate an extendableterminus. The label corresponds to the probe family to which the probebelongs.

Successive cycles of extension, ligation, and detection produce anordered list of probe families to which successive successfully ligatedprobes belong. The ordered list of probe families is used to obtaininformation about the sequence. However, knowing to which probe family anewly ligated probe belongs is not by itself sufficient to determine theidentity of a nucleotide in the template. Instead, knowing to whichprobe family the newly ligated probe belongs eliminates certainsequences as possibilities for the sequence of the constrained portionof the probe but leaves at least two possibilities for the identity ofthe nucleotide at each position. Thus there are at least twopossibilities for the identity of the nucleotides in the template thatare located at opposite positions to the nucleotides in the constrainedportion of the newly ligated probe (i.e., the nucleotides that arecomplementary to the nucleotides in the constrained portion of theprobe).

In certain embodiments, after performing a desired number of cycles, aset of candidate sequences is generated using the ordered series ofprobe family identities. The set of candidate sequences may providesufficient information to achieve an objective. In preferred embodimentsof the invention one or more additional steps are performed to selectthe correct sequence from among the candidate sequences. For example,the sequences can be compared with a database of known sequences, andthe candidate sequence closest to one of the sequences in the databaseis selected as the correct sequence. In other embodiments the templateis subjected to another round of sequencing by successive cycles ofextension, ligation, detection, and cleavage, using a differentlyencoded set of probe families, and the information obtained in thesecond round is used to select the correct sequence. In otherembodiments at least one item of information is combined with theinformation obtained from ordered list of probe family identities todetermine the sequence.

The invention also provides methods of performing error checking whentemplates are sequenced using probe families. Certain of the methodsdistinguish between single nucleotide polymorphisms (SNPs) andsequencing errors.

The invention also provides nucleic acid fragments (e.g., DNA fragments)containing at least two segments of interest (e.g., at least two tags)and at least three primer binding regions (PBRs), such that at least twodistinct templates, each corresponding to a segment of interest, can beamplified from each fragment. A “primer binding region” is a portion ofa nucleic acid to which an oligonucleotide can hybridize such that theoligonucleotide can serve as an amplification primer, sequencing primer,initializing oligonucleotide, etc. Thus the primer binding region shouldhave a known sequence in order to allow selection of a suitablecomplementary olignucleotide. As used herein and in the figures, aportion of a nucleic acid strand used in a method of the invention maybe referred to as a primer binding region regardless of whether, in thepractice of the method, the primer actually binds to the region or bindsto the corresponding portion of a complementary strand of the nucleicacid strand. Thus a portion of a nucleic acid may be referred to as aprimer binding region regardless of whether, when used in a method ofthe invention, a primer actually binds to that region (in which case thesequence of the primer is complementary or substantially complementaryto that of the region) or binds to the complement of the region (inwhich case the sequence of the primer is identical to or substantiallyidentical to the sequence of the primer binding region) A segment ofinterest is any segment of nucleic acid for which sequence informationis desired. For example, a sequence of interest may be a tag, and forpurposes of the present disclosure it will be assumed that the segmentof interest is a tag (also referred to herein and elsewhere as an “endtag”). However, it is to be understood that the invention is not limitedto segments of interest that are tags. In certain embodiments the atleast two tags are a paired tag. The nucleic acid fragments can containone or more pairs of tags, e.g., one or more paired tags, e.g., 2, 3, 4,5, or more pairs of paired tags. The invention further provideslibraries containing such nucleic acid fragments, and methods for makingthe templates and libraries.

The invention further provides a microparticle, e.g., a bead, having atleast two distinct populations of nucleic acids attached thereto,wherein each of the at least two populations consists of a plurality ofsubstantially identical nucleic acids, and wherein the populations wereproduced by amplification (e.g., PCR amplification) from a singlenucleic acid fragment. In some embodiments the single nucleic acidfragment contains a 5′ tag and 3′ tag, wherein the 5′ and 3′ tags are apaired tag. In some embodiments in which the single nucleic acidfragment contains a 5′ tag and a 3′ tag of a pair, one of thepopulations of nucleic acids attached to the microparticle comprises atleast a portion of the 5′ tag and one of the populations of nucleicacids attached to the microparticle comprises at least a portion of the3′ tag. In preferred embodiments one of the populations comprises acomplete 5′ tag and one of the populations comprises a complete 3′ tag.

The nucleic acid fragment contains multiple PBRs, at least one of whichis located between the tags and at least two of which flank a portion ofthe nucleic acid fragment that contains the tags, so that a regioncomprising at least a portion of the 5′ tag can be amplified, and aregion comprising at least a portion of the 3′ tag can be amplified, toproduce two distinct populations of nucleic acids. In preferredembodiments the entire 5′ tag and the entire 3′ tag can be amplified.For example, the nucleic acid fragment can contain first and secondprimer binding sites flanking the 5′ tag and also third and fourthprimer binding sites flanking the 3′ tag. A PCR amplification usingprimers that bind to the first and second primer binding sites amplifiesthe 5′ tag. A PCR amplification using primers that bind to the third andfourth primer binding sites amplifies the 3′ tag. It will be appreciatedthat the primers should be selected so that extension from each primerproceeds towards the region of the DNA fragment containing the tag to beamplified. Alternately, a first primer binding site can be locatedupstream of one of the tags, and a second primer binding site can belocated downstream of the other tag, and a third primer binding site canbe located between the two tags. The third primer binding site serves asa binding site for a forward primer for a PCR amplification thatamplifies one of the tags and serves as a binding site for a reverseprimer for a PCR amplification that amplifies the other tag. Thus in oneembodiment the invention provides a microparticle, e.g., a bead, havingat least two distinct populations of nucleic acids attached thereto,wherein each of the at least two populations consists of a plurality ofsubstantially identical nucleic acids, and wherein a first distinctpopulation comprises a 5′ tag and a second distinct population comprisesa 3′ tag.

The invention further provides a population of microparticles, e.g.,beads, wherein individual microparticles having at least two distinctpopulations of nucleic acids attached thereto, wherein each of the atleast two populations consists of a plurality of substantially identicalnucleic acids, and wherein the populations were produced byamplification (e.g., PCR amplification) from a single DNA fragment. Thesubstantially identical populations can be, e.g., a 5′ tag and a 3′ tag.The invention further provides arrays of such microparticles and methodsof sequencing that involve sequencing the populations of substantiallyidentical nucleic acids. For example, in one embodiment, each of the twopopulations of substantially identical nucleic acids attached to anindividual microparticle comprises a different primer binding region(PBR), so that by using different sequencing primers, one of thepopulations can be sequenced without interference from the otherpopulation. If more than two substantially identical populations ofsubstantially identical nucleic acids are attached to a singlemicroparticle, each of the populations can have a unique PBR, such thata primer that binds to a given PBR does not bind to a PBR present in theother substantially identical populations of nucleic acids attached tothe microparticle. Thus the methods of the invention allow for producingmicroparticles having at least two different substantially identicalpopulations of nucleic acids attached thereto (e.g., a multiple copiesof template containing a 5′ tag and multiple copies of templatecontaining a 3′ tag), wherein the tags are paired tags. In accordancewith the inventive methods, the templates contain different PBRs, whichprovide binding sites for sequencing primers. Therefore, by selecting asequencing primer complementary to the PBR in the template that containsthe 5′ tag, sequence information can be obtained from the 5′ tag withoutinterference from the template containing the 3′ tag, even though thetemplate containing the 3′ tag is also present on the samemicroparticle. By selecting a sequencing primer complementary to the PBRin the template that contains the 3′ tag, sequence information can beobtained from the 3′ tag without interferene from the templatecontaining the 5′ tag, even though the template containing the 5′ tag isalso present on the same microparticle. The fact that both of the pairedtags are present on the same microparticle means that the sequence ofthe 5′ and 3′ paired tags can be associated with one another, just aswould be the case if they were present within a single template as inthe prior art.

The invention also provides automated sequencing systems that may beused, e.g., to sequence templates arrayed in or on a substantiallyplanar support. The invention further provides image processing methods,which may be stored on a computer-readable medium such as a hard disc,CD, zip disk, flash memory, or the like. In certain preferredembodiments the system achieves 40,000 nucleotide identifications persecond, or more. In certain preferred embodiments the system generates8.6 gigabytes (Gb) of sequence data per day (24 hours), or more. Incertain preferred embodiments the system produces 48 Gb of sequenceinformation (nucleotide identifications) per day, or more.

In addition, the invention provides a computer-readable medium thatstores information generated by applying the inventive sequencingmethods. The information may be stored in a database.

This application refers to various patents, patent applications, journalarticles, and other publications, all of which are incorporated hereinby reference. In addition, the following standard reference works areincorporated herein by reference: Current Protocols in MolecularBiology, John Wiley & Sons, N.Y., edition as of July 2002; Sambrook,Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3^(rd)ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. Inthe event of a conflict between the instant specification and anydocument incorporated by reference, the specification shall control, itbeing understood that the determination of whether a conflict orinconsistency exists is within the discretion of the inventors and canbe made at any time.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A diagramatically illustrates initialization followed by twocycles of extension, ligation, and identification.

FIG. 1B diagramatically illustrates initialization followed by twocycles of extension, ligation, and identification in an embodiment inwhich extension proceeds inwards from the free end of the templatetowards a support.

FIG. 2 shows a scheme for assigning colors to oligonucleotide probes inwhich the identity of the 3′ base of the probe is determined byidentifying the color of a fluorophore.

FIG. 3A diagramatically shows extended duplexes resulting fromhybridization of initializing oligonucleotides at different positions inthe binding region of a template followed by ligation of extensionprobes.

FIG. 3B diagrammatically shows assembly of a continuous sequence (listedin the Sequence Listing as SEQ ID NO. 7) by using the extension,ligation, and cleavage method with extension probes designed to readevery 6^(th) base of the template molecule.

FIG. 4A illustrates a 5′-S-phosphorothiolate linkage (3′-O—P—S-5′).

FIG. 4B illustrates a 3′-S-phosphorothiolate linkage (3′-S—P—O-5′).

FIG. 5A diagramatically illustrates a single cycle of extension,ligation, and cleavage for sequencing in the 5′→3′ direction usingextension probes having 3′-O—P—S-5′ phosphorothiolate linkages.

FIG. 5B diagramatically illustrates a single cycle of extension,ligation, and cleavage for sequencing in the 3′→5′ direction usingextension probes having 3′-S—P—O-5′ phosphorothiolate linkages.

FIG. 6A-6F is a more detailed diagrammatic illustration of severalsequencing reactions performed on a single template. The reactionsutilize initializing oligonucleotides that bind to different portions ofthe template. The sequence of the extended strand depicted in FIG. 6D islisted as SEQ ID NO. 8. The sequence of the extended strand depicted inFIG. 6E is listed as SEQ ID NO. 9. The sequence of the extended stranddepicted in FIG. 6F is listed as SEQ ID NO. 10. The sequence depicted inFIG. 6F corresponding to the template is listed as SEQ ID NO. 11.

FIG. 7 is a schematic showing a synthesis scheme for3′-phosphoroamidites of dA and dG.

FIG. 8A-8E shows results of a gel shift assay demonstrating two cyclesof successful ligation and cleavage of extension probes containingphosphorothiolate linkages. The sequence of the initializingoligonucleotide is listed as SEQ ID NO. 12. The sequence of the LST7template is listed as SEQ ID NO. 13. The sequence depicted under thelabel “Ligation 2” is listed as SEQ ID NO. 14.

FIG. 8F shows a schematic diagram of the mechanism of ligation by DNAligases.

FIG. 9 shows results of a gel shift assay demonstrating the ligationefficiency of degenerate inosine-containing oligonucleotide probes. Thesequences of the primer and LST1 template are listed as SEQ ID NOs. 15and 16 respectively.

FIG. 10A and FIG. 10B shows results of a gel shift assay demonstratingthe ligation efficiency of degenerate inosine-containing oligonucleotideprobes on multiple templates.

FIG. 11A-11C shows results of an analysis conducted to assess thefidelity of each of two DNA ligases (T4 DNA ligase (FIG. 11(C)) and TaqDNA ligase (FIG. 11(B))) for 3′→5′ extensions. The sequences of theprimer and LST1 template (FIG. 11(A)) are listed as SEQ ID NOs. 17 and18 respectively.

FIG. 12 shows results of a gel shift assay (A) demonstrating theligation efficiency of degenerate inosine-containing oligonucleotideprobes and of a direct sequencing analysis of the ligation reactions (B)conducted to assess the fidelity of T4 DNA ligase in oligonucleotideprobe ligation. The sequences of the primer and LST6 template are listedas SEQ ID NOs. 15 and 19, respectively. The sequences depicted in FIG.12(B), from top to bottom, are listed as SEQ ID NOs. 20-25.

FIG. 13A-13C shows results of an experiment that demonstrates in-gelligation when bead-based templates are embedded in polyacrylamide gelson slides. FIG. 13(A) shows a schematic of the ligation reaction. In gelligation reactions were performed in the absence FIG. 13(B) and in thepresence FIG. 13(C) of T4 DNA ligase. The sequences of the primer andLST1 template are listed as SEQ ID NOs. 15 and 26 respectively.

FIG. 14A shows an image of an emulsion PCR reaction performed on beadshaving attached first amplification primers, using a fluorescentlylabeled second amplification primer and an excess of template.

FIG. 14B (top) shows a fluorescence image of a portion of a slide onwhich beads with an attached template, to which a Cy3-labeledoligonucleotide was hybridized, were immobilized within a polyacrylamidegel. (This slide was used in a different experiment, but isrepresentative of the slides used here.) FIG. 14B (bottom) shows aschematic diagram of a slide equipped with a Teflon mask to enclose thepolyacrylamide solution.

FIG. 15 illustrates three sets of labeled oligonucleotide probesdesigned to address issues of probe specificity and selectivity and alsoshows excitation and emission values for a set of four spectrallyresolvable labels.

FIG. 16A-16D shows results of an experiment confirming 4-color spectralidentity of oligonucleotide probes. Slides containing four uniquesingle-stranded template populations FIG. 16(A) were subjected tohybridization and ligation reactions using an oligonucleotide probemixture that contained four unique fluorophore probes, were imaged underbright light FIG. 16(B) and with fluorescence excitation using fourbandpass filters before and after ligation. Individual populations werepseudocolored FIG. 16(C). The spectral identity, which showed minimalsignal overlap, is plotted in FIG. 16(D). The sequences of theinitializing oligonucleotide and LST1 template are listed as SEQ ID NOs.15 and 26 respectively. The sequences of the extended initializingoligonucleotides with FAM-, Cy5-, CAL560-, and CAL610-labeled probes arelisted as SEQ ID NOs. 27, 28, 30, and 32 respectively. The sequences ofthe LST2, LST3, and LST4 templates are listed as SEQ ID NOs. 29, 31, and33 respectively.

FIG. 17A-17D shows results from an experiment confirming ligationspecificity of oligonucleotide extension probes. FIG. 17(A) shows aschematic outline of ligation. FIG. 17(B) is a bright light image, andFIG. 17(C) is a corresponding fluorescence image of a population ofbeads embedded in a polyacrylamide gel after ligation. FIG. 17(D) showsfluorescence detected from each label before (pre) or after (post)ligation. The sequences of the extended initializing oligonucleotidedwith probe and LST1 template are listed as SEQ ID NOs. 27 and 26respectively.

FIG. 18A-18D shows results from another experiment confirming ligationspecificity and selectivity of oligonucleotide extension probes. FIG.18(A) shows a schematic outline of ligation. FIG. 18(B) is a brightlight image, and FIG. 18(C) is a corresponding fluorescence image of apopulation of beads embedded in a polyacrylamide gel after ligation.FIG. 18(D) shows expected versus observed ligation frequencies, showinga high correlation between frequencies expected based on the proportionof particular extension probes in a population and frequencies observed.The sequences of the initializing oligonucleotide and LST1 template arelisted as SEQ ID NOs. 15 and 26 respectively. The sequences of theextended initializing oligonucleotides with CAL560-, CAL610-, Cy5-, andFAM-labeled probes are listed as SEQ ID NOs. 34, 36, 38, and 27respectively. The sequences of the LST1.A, LST1.G, LST1.C, and LST1.Ttemplates are listed as SEQ ID NOs. 35, 37, 39 and 26 respectively.

FIG. 19A-19E shows results from an experiment confirming that degenerateand universal base containing oligonucleotide extension probe pools canbe used to afford specific and selective in-gel ligation. FIG. 19(A)shows a schematic outline of the ligation experiment, illustrating fourdifferentially labeled degenerate inosine-containing probe poolsfollowing ligation. FIG. 19(B) is a bright light image, and FIG. 19(C)is a corresponding fluorescence image of a population of beads embeddedin a polyacrylamide gel after ligation. FIG. 19(D) shows expected versusobserved ligation frequencies, showing a high correlation betweenfrequencies expected based on the proportion of particular extensionprobes in a population and frequencies observed. FIG. 19(E) shows ascatter plot of the raw unprocessed data and filtered data representingthe top 90% of bead signal values. The sequences of the initializingoligonucleotide and LST1 template are listed as SEQ ID NOs. 15 and 26respectively. The sequences of the extended initializingoligonucleotides with FA-, Cy-, Cal61-, and Cal56-labeled probes arelisted as SEQ ID NOs. 40, 41, 43, and 45 respectively. The sequences ofthe LST2, LST3, and LST4 templates are listed as SEQ ID NOs. 42, 44, and46 respectively.

FIG. 20 is a chart showing the signal detected in sequential cycles ofhybridization and stripping of an initializing oligonucleotide (primer)to a template. As shown in the figure, minimal signal loss occurred over10 cycles.

FIG. 21 is a photograph of an automated sequencing system that may beused to gather sequence information, e.g., from templates arrayed in oron a substantially planar support. Also shown is a dedicated computerfor controlling operation of various components of the system,processing and storing collected image data, providing a user interface,etc. The lower portion of the figure shows an enlarged view of a flowcell oriented to achieve gravimetric bubble displacement.

FIG. 22 shows a schematic diagram of a high throughput automatedsequencing instrument that may be used to sequence templates arrayed inor on a substantially planar support.

FIG. 23 shows a scatter plot of alignment inconsistency, illustratingminimal inconsistency over 30 frames.

FIGS. 24A-24I shows schematic diagrams of inventive flow cells orportions thereof in a variety of different views.

FIG. 25A shows an exemplary encoding for a preferred collection of probefamilies comprising partially constrained probes comprising constrainedportions that are 2 nucleotides in length.

FIG. 25B shows a preferred collection of probe families (upper panel)and a cycle of ligation, detection, and cleavage (lower panel).

FIG. 26 shows an exemplary encoding for another preferred collection ofprobe families comprising partially constrained probes comprisingconstrained portions that are 2 nucleotides in length.

FIGS. 27A-27C represent an alternate method to schematically define the24 preferred collections of probe families that are defined in Table 1.

FIG. 28 shows a less preferred collection of probe families in which theprobes comprise constrained portions that are 2 nucleotides in length.

FIG. 29A shows a diagram that can be used to generate constrainedportions for a collection of probe families that comprises probes with aconstrained portion 3 nucleotides long.

FIG. 29B shows a diagram a mapping scheme that can be used to generateconstrained portions for a collection of probe families that comprisesprobes with a constrained portion 3 nucleotides long from the 24preferred collections of probe families.

FIG. 30 shows a method in which sequence determination is performedusing a collection of probe families. An embodiment using a preferredset of probe families is depicted.

FIGS. 31A-31C show a method in which sequence determination is performedusing a first collection of probe families to generate candidatesequences and a second collection of probe families to decode.

FIG. 32 shows a method in which sequence determination is performedusing a less preferred collection of probe families.

FIG. 33A shows a schematic diagram of a slide with beads attachedthereto. DNA templates are attached to the beads.

FIG. 33B shows a population of beads attached to a slide. The lowerpanels show the same region of the slide under white light (left) andfluorescence microscopy. The upper panel shows a range of beaddensities.

FIGS. 34A-34C show a scheme for amplifying both tags of a paired tagpresent in a nucleic acid fragment (template) as individual populationsof nucleic acids and capturing them to a microparticle via theamplification process.

FIGS. 35A and 35B show details of primer design and amplification forthe scheme of FIG. 35. Both strands of a nucleic acid fragment(template) are shown for clarity. Primers and primer binding regionshaving the same sequence are presented in the same color. For example,P1 is represented in dark blue, indicating that primer P1, which ispresent on the microparticle and in solution, has the same sequence asthe correspondingly colored portion of the indicated strand of thetemplate. The dark blue region of the template, labeled P1, may bereferred to as a primer binding region even though the correspondingprimer (P1) in fact binds to the complementary portion of the otherstrand and has the same sequence as primer P1.

FIGS. 35C and 35D show sequencing of the first and second tags,respectively, attached to a microparticle produced by the method ofFIGS. 35A and 35B.

DEFINITIONS

To facilitate understanding of the description of the invention, thefollowing definitions are provided. It is to be understood that, ingeneral, terms not otherwise defined are to be given their meaning ormeanings as generally accepted in the art.

As used herein, an “abasic residue” is a residue that has the structureof the portion of a nucleoside or nucleotide that remains after removalof the nitrogenous base or removal of a sufficient portion of thenitrogenous base such that the resulting molecule no longer participatesin hydrogen bonds characteristic of a nucleoside or nucleotide. Anabasic residue may be generated by removing a nitrogenous base from anucleoside or nucleotide. However, the term “abasic” is used to refer tothe structural features of the residue and is independent of the mannerin which the residue is produced. The terms “abasic residue” and “abasicsite” are used herein to refer to a residue within a nucleic acid thatlacks a purine or pyrimidine base.

An “apurinic/apyrimidinic (AP) endonuclease”, as used herein, refers toan enzyme that cleaves a bond on either the 5′ side, the 3′ side, orboth the 5′ and 3′ sides of an abasic residue in a polynucleotide. Incertain embodiments of the invention the AP endonuclease is an AP lyase.Examples of AP endonucleases include, but are not limited to, E. coliendonuclease VIII and homologs thereof and E. coli endonuclease III andhomologs thereof. It is to be understood that references to specificenzymes, e.g., endonucleases such as E. coli Endo VIII, Endo V, etc.,are intended to encompass homologs from other species that arerecognized in the art as being homologs and as possessing similarbiochemical activity with respect to removal of damaged bases and/orcleavage of DNA containing abasic residues or other trigger residues.

As used herein, the term “array” refers to a collection of entities thatis distributed over or in a support matrix; preferably, individualentities are spaced at a distance from one another sufficient to permitthe identification of discrete features of the array by any of a varietyof techniques. The entities may be, for example, nucleic acid molecules,clonal populations of nucleic acid molecules, microparticles (optionallyhaving clonal populations of nucleic acid molecules attached thereto),etc. When used as a verb, the term “array” and variations thereof refersto any process for forming an array, e.g., distributing entities over orin a support matrix.

A “damaged base” is a purine or pyrimidine base that differs from an A,G, C, or T in such a manner as to render it a substrate for removal fromDNA by a DNA glycosylase. Uracil is considered a damaged base forpurposes of the present invention. In some embodiments of the inventionthe damaged base is hypoxanthine.

“Degenerate”, with respect to a position in a polynucleotide that is oneof a population of polynucleotides, means that the identity of the basethat forms part of the nucleoside occupying that position varies amongdifferent members of the population. Thus the population containsindividual members whose sequence differs at the degenerate position.The term “position” refers to a numerical value that is assigned to eachnucleoside in a polynucleotide, generally with respect to the 5′ or 3′end. For example, the nucleoside at the 3′ end of an extension probe maybe assigned position 1. Thus in a pool of extension probes of structure3′-XXXNXXXX-5′, the N is at position 4. Position 4 is considereddegenerate if, in different members of the pool, the identity of N canvary. The pool of extension probes is also said to be degenerate atposition N. A position is said to be k-fold degenerate if it can beoccupied by nucleosides having any of k different identities. Forexample, a position that can be occupied by nucleosides comprisingeither of 2 different bases is 2-fold degenerate.

“Determining information about a sequence” encompasses “sequencedetermination” and also encompasses other levels of information such aseliminating one or more possibilities for the sequence. It is noted thatperforming sequence determination on a polynucleotide typically yieldsequivalent information regarding the sequence of a perfectlycomplementary (100% complementary) polynucleotide and thus is equivalentto sequence determination performed directly on a perfectlycomplementary polynucleotide.

“Independent”, with respect to a plurality of elements, e.g.,nucleosides in an oligonucleotide probe molecule or portion thereof,means that the identity of each element does not limit and is notlimited by the identity of any of the other elements, e.g., the identityof each element is selected without regard for the identity of any ofthe other element(s). Thus knowing the identity of one or more of theelements does not provide any information regarding the identity of anyof the other elements. For example, the nucleosides in the sequence NNNNare independent if the identity of each N can be A, G, C, or T,regardless of the identity of any other N.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically.

The term “microparticle” is used herein to refer to particles having asmallest cross-sectional dimension of 50 microns or less, preferably 10microns or less. In certain embodiments the smallest cross-sectionaldimension is approximately 3 microns or less, approximately 1 micron orless, approximately 0.5 microns or less, e.g., approximately 0.1, 0.2,0.3, or 0.4 microns. Microparticles may be made of a variety ofinorganic or organic materials including, but not limited to, glass(e.g., controlled pore glass), silica, zirconia, cross-linkedpolystyrene, polyacrylate, polymehtymethacrylate, titanium dioxide,latex, polystyrene, etc. See, e.g., U.S. Pat. No. 6,406,848, for varioussuitable materials and other considerations. Dyna beads, available fromDynal, Oslo, Norway, are an example of commercially availablemicroparticles of use in the present invention. Magnetically responsivemicroparticles can be used. The magnetic responsiveness of certainpreferred microparticles permits facile collection and concentration ofthe microparticle-attached templates after amplification, andfacilitates additional steps (e.g., washes, reagent removal, etc.). Incertain embodiments of the invention a population of microparticleshaving different shapes (e.g., some spherical and others nonspherical)is employed.

The term “microsphere” or “bead” is used herein to refer tosubstantially spherical microparticles having a diameter of 50 micronsor less, preferably 10 microns or less. In certain embodiments thediameter is approximately 3 microns or less, approximately 1 micron orless, approximately 0.5 microns or less, e.g., approximately 0.1, 0.2,0.3, or 0.4 microns. In certain embodiments of the invention apopulation of monodisperse microspheres is used, i.e., the microspheresare of substantially uniform size. For example, the diameters of themicroparticles may have a coefficient of variation of less than 5%,e.g., 2% of less, 1% or less, etc. However, in other embodiments thecoefficient of variation of a population of microparticles is 5% orgreater, e.g., 5%, between 5% and 10% (inclusive), between 10% and 25%,inclusive, etc. In certain embodiments a mixed population ofmicroparticles is used. For example, a mixture of two populations, eachof which has a coefficient of variation of less than 5%, may be used,resulting in a mixed population that is not monodisperse. As an example,a mixture of microspheres having diameters of 1 micron and 3 microns canbe employed. In certain embodiments of the invention additionalinformation is provided by the size of the microsphere when sequencingis performed using templates attached to microspheres of a populationthat is not monodisperse. For example, different libraries of templatesmay be attached to differently sized microspheres. Also, since fewertemplate molecules may be attached to smaller particles, the intensityof the signals may vary, which may facilitate multiplex sequencing.

The term “nucleic acid sequence” as used herein can refer to the nucleicacid material itself and is not restricted to the sequence information(i.e. the succession of letters chosen among the five base letters A, G,C, T, or U) that biochemically characterizes a specific nucleic acid,e.g., a DNA or RNA molecule. Nucleic acids shown herein are presented ina 5′→3′ orientation unless otherwise indicated.

A “nucleoside” comprises a nitrogenous base linked to a sugar molecule.As used herein, the term includes natural nucleosides in their 2′-deoxyand 2′-hydroxyl forms as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992) and nucleosideanalogs. For example, natural nucleosides include adenosine, thymidine,guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine. Nucleoside “analogs” refers tosynthetic nucleosides having modified base moieties and/or modifiedsugar moieties, e.g. described generally by Scheit, Nucleotide Analogs(John Wiley, New York, 1980). Such analogs include synthetic nucleosidesdesigned to enhance binding properties, reduce degeneracy, increasespecificity, and the like. Nucleoside analogs include 2-aminoadenosine,2-thiothymidine, pyrrolo-pyrimidine, 3-methyl adenosine,C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,2-thiocytidine, etc. Nucleoside analogs may comprise any of theuniversal bases mentioned herein.

The term “organism” is used herein to indicate any living or nonlivingentity that comprises nucleic acid that is capable of being replicatedand is of interest for sequence determination. It includes plasmids;viruses; prokaryotic, archaebacterial and eukaryotic cells, cell lines,fungi, protozoa, plants, animals, etc.

“Perfectly matched duplex” in reference to the protruding strands ofprobes and template polynucleotides means that the protruding strandfrom one forms a double stranded structure with the other such that eachnucleoside in the double stranded structure undergoes Watson-Crickbasepairing with a nucleoside on the opposite strand. The term alsocomprehends the pairing of nucleoside analogs, such as deoxyinosine,nucleosides with 2-aminopurine bases, and the like, that may be employedto reduce the degeneracy of the probes, whether or not such pairinginvolves formation of hydrogen bonds.

The term “plurality” means more than one.

The term “polymorphism” is given its ordinary meaning in the art andrefers to a difference in genome sequence among individuals of the samespecies. A “single nucleotide polymorphism” (SNP) refers to apolymorphism at a single position.

“Polynucleotide”, “nucleic acid”, or “oligonucleotide” refers to alinear polymer of nucleosides (including deoxyribonucleosides,ribonucleosides, or analogs thereof) joined by internucleosidiclinkages. Typically, a polynucleotide comprises at least threenucleosides. In certain embodiments of the invention one or morenucleosides in an extension probe comprises a universal base. Usuallyoligonucleotides range in size from a few monomeric units, e.g. 3-4, toseveral hundreds of monomeric units. Whenever a polynucleotide such asan oligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,unless otherwise noted. The letters A, C, G, and T may be used to referto the bases themselves, to nucleosides, or to nucleotides comprisingthe bases, as is standard in the art.

In naturally occurring polynucleotides, the internucleoside linkage istypically a phosphodiester bond, and the subunits are referred to as“nucleotides”. However, oligonucleotide probes comprising otherinternucleoside linkages, such as phosphorothiolate linkages, are usedin certain embodiments of the invention. It will be appreciated that oneor more of the subunits that make up such an oligonucleotide probe witha non-phosphodiester linkage may not comprise a phosphate group. Suchanalogs of nucleotides are considered to fall within the scope of theterm “nucleotide” as used herein, and nucleic acids comprising one ormore internucleoside linkages that are not phosphodiester linkages arestill referred to as “polynucleotides”, “oligonucleotides”, etc. Inother embodiments, a polynucleotide such as an oligonucleotide probecomprises a linkage that contains an AP endonuclease sensitive site. Forexample, the oligonucleotide probe may contain an abasic residue, aresidue containing a damaged base that is a substrate for removal by aDNA glycosylase, or another residue or linkage that is a substrate forcleavage by an AP endonuclease. In another embodiment an oligonucleotideprobe contains a disaccharide nucleoside.

The term “primer” refers to a short polynucleotide, typically betweenabout 10-100 nucleotides in length, that binds to a targetpolynucleotide or “template” by hybridizing with the target. The primerpreferably provides a point of initiation for template-directedsynthesis of a polynucleotide complementary to the target, which cantake place in the presence of appropriate enzyme(s), cofactors,substrates such as nucleotides, oligonucleotides, etc. The primertypically provides a terminus from which extension can occur. In thecase of primers for synthesis catalyzed by a polymerase enzyme such as aDNA polymerase (e.g., in “sequencing by synthesis”, polymerase chainreaction (PCR) amplification, etc.), the primer typically has, or can bemodified to have, a free 3′ OH group. Typically a PCR reaction employs apair of primers (first and second amplification primers) including an“upstream” (or “forward”) primer and a “downstream” (or “reverse”)primer, which delimit a region to be amplified. In the case of primersfor synthesis by successive cycles of extension, ligation (andoptionally cleavage), the primer typically has, or can be modified tohave, a free 5′ phosphate group or 3′ OH group that serves as asubstrate for DNA ligase.

As used herein, a “probe family” refers to a group of probes, each ofwhich comprises the same label.

As used herein “sequence determination”, “determining a nucleotidesequence”, “sequencing”, and like terms, in reference to polynucleotidesincludes determination of partial as well as full sequence informationof the polynucleotide. That is, the term includes sequence comparisons,fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofeach nucleoside of the target polynucleotide within a region ofinterest. In certain embodiments of the invention “sequencedetermination” comprises identifying a single nucleotide, while in otherembodiments more than one nucleotide is identified. In certainembodiments of the invention, sequence information that is insufficientby itself to identify any nucleotide in a single cycle is gathered.Identification of nucleosides, nucleotides, and/or bases are consideredequivalent herein. It is noted that performing sequence determination ona polynucleotide typically yields equivalent information regarding thesequence of a perfectly complementary (100% complementary)polynucleotide and thus is equivalent to sequence determinationperformed directly on a perfectly complementary polynucleotide.

“Sequencing reaction” as used herein refers to a set of cycles ofextension, ligation, and detection. When an extended duplex is removedfrom a template and a second set of cycles is performed on the template,each set of cycles is considered a separate sequencing reaction thoughthe resulting sequence information may be combined to generate a singlesequence.

“Semi-solid”, as used herein, refers to a compressible matrix with botha solid and a liquid component, wherein the liquid occupies pores,spaces or other interstices between the solid matrix elements. Exemplarysemi-solid matrices include matrices made of polyacrylamide, cellulose,polyamide (nylon), and cross-linked agarose, dextran and polyethyleneglycol. A semi-solid support may be provided on a second support, e.g.,a substantially planar, rigid support, also referred to as a substrate,which supports the semi-solid support.

“Support”, as used herein, refers to a matrix on or in which nucleicacid molecules, microparticles, and the like may be immobilized, i.e.,to which they may be covalently or noncovalently attached or, in or onwhich they may be partially or completely embedded so that they arelargely or entirely prevented from diffusing freely or moving withrespect to one another.

A “trigger residue” is a residue that, when present in a nucleic acid,renders the nucleic acid more susceptible to cleavage (e.g., cleavage ofthe nucleic acid backbone) by a cleavage agent (e.g., an enzyme, silvernitrate, etc.) or combination of agents than would be an otherwiseidentical nucleic acid not including the trigger residue, and/or issusceptible to modification to generate a residue that renders thenucleic acid more susceptible to such cleavage. Thus presence of atrigger residue in a nucleic acid can result in presence of a scissilelinkage in the nucleic acid. For example, an abasic residue is a triggerresidue since the presence of an abasic residue in a nucleic acidrenders the nucleic acid susceptible to cleavage by an enzyme such as anAP endonuclease. A nucleoside containing a damaged base is a triggerresidue since the presence of a nucleoside comprising a damaged base ina nucleic acid also renders the nucleic acid more susceptible tocleavage by an enzyme such as an AP endonuclease, e.g., after removal ofthe damaged base by a DNA glycosylase. The cleavage site may be at abond between the trigger residue and an adjacent residue or may be at abond that is one or more residues removed from the trigger residue. Forexample, deoxyinosine is a trigger residue since the presence of adeoxyinosine in a nucleic acid renders the nucleic acid more susceptibleto cleavage by E. coli Endonuclease V and homologs thereof. Such enzymescleave the second phosphodiester bond 3′ to deoxyinosine. Any of theprobes disclosed herein may contain one or more trigger residues. Thetrigger residue may, but need not, comprise a ribose or deoxyribosemoiety. Preferably the cleavage agent is one that does not substantiallycleave a nucleic acid in the absence of a trigger residue but exhibitssignificant cleavage activity against a nucleic acid that contains thetrigger residue under the same conditions, which conditions may includethe presence of agents that modify the nucleic acid to render itsensitive to the cleavage agent. For example, preferably if the cleavageagent is present in a composition containing nucleic acids that areidentical in length and composition except that one of them contains thetrigger residue and the other of them does not contain the triggerresidue, the likelihood that the nucleic acid containing the triggerresidue will be cleaved is at least: 10; 25; 50; 100; 250; 500; 1000;2500; 5000; 10,000; 25,000; 50,000; 100,000; 250,000; 500,000; 1,000,000or more, as great as the likelihood that the nucleic acid not containingthe trigger residue will be cleaved, e.g., the ratio of the likelihoodof cleavage of a nucleic acid containing a trigger residue to thelikelihood of cleavage of a nucleic acid not containing the triggerresidue but otherwise identical is between 10 and 10⁶, or any integralsubrange thereof. It will be appreciated that the ratio may differdepending upon the particular nucleic acid and location and nucleotidecontext of the trigger residue.

Preferably if the nucleic acid containing the trigger residue needs tobe modified in order to render the nucleic acid susceptible to cleavageby a cleavage agent, such modification occurs readily in the presence ofsuitable modifying agent(s), e.g., the modification occurs in reasonableyield and in a reasonable period of time. For example, in certainembodiments of the invention at least 50%, at least 60%, at least 70%,preferably at least 80%, at least 90% or more preferably at least 95% ofthe nucleic acids containing the trigger residue are modified within,e.g., 24 hours, preferably within 12 hours, more preferably within lessthan 1 minute to 4 hours.

A variety of suitable trigger residues and corresponding cleavagereagents are exemplified herein. Any trigger residue and cleavagereagent having similar activity to those described herein may be used.One of ordinary skill in the art will be able to determine whether aparticular trigger residue and cleavage reagent combination is suitablefor use in the present invention, e.g., whether the cleavage efficiencyand speed, the selectivity of the cleavage agent for nucleic acidscontaining a trigger residue, etc, are suitable for use in the methodsof the invention. Note that a “trigger residue” is distinguished from anucleotide that simply forms part of a restriction enzyme site in thatthe ability of the trigger residue to confer increased susceptibility tocleavage does not, in general, depend significantly on the particularsequence context in which the trigger residue is found although, asnoted above, the context can have some influence on the susceptibilityto modification and/or cleavage. Of course depending on the surroundingnucleotides, a trigger residue may form part of a restriction site.Thus, in most cases, the cleavage agent is not a restriction enzyme,though use of an enzyme that is both a restriction enzyme and hasnon-sequence specific cleavage ability is not excluded.

A “universal base”, as used herein, is a base that can “pair” with morethan one of the bases typically found in naturally occurring nucleicacids and can thus substitute for such naturally occurring bases in aduplex. The base need not be capable of pairing with each of thenaturally occurring bases. For example, certain bases pair only orselectively with purines, or only or selectively with pyrimidines.Certain preferred universal bases (fully universal bases) can pair withany of the bases typically found in naturally occurring nucleic acidsand can thus substitute for any of these bases in duplex. The base neednot be equally capable of pairing with each of the naturally occurringbases. If a probe mix contains probes that comprise (at one or morepositions) a universal base that does not pair with all of the naturallyoccurring nucleotides, it may be desirable to utilize two or moreuniversal bases at that position in the particular probe so that atleast one of the universal bases pairs with A, at least one of theuniversal bases pairs with G, at least one of the universal bases pairswith C, and at least one of the universal bases pairs with T.

A number of universal bases are known in the art including, but notlimited to, hypoxanthine, 3-nitropyrrole, 4-nitroindole, 5-nitroindole,4-nitrobenzimidazole, 5-nitroindazole, 8-aza-7-deazaadenine,6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P. Kong Thoo Lin. andD. M. Brown, Nucleic Acids Res., 1989, 17, 10373-10383),2-amino-6-methoxyaminopurine (D. M. Brown and P. Kong Thoo Lin,Carbohydrate Research, 1991, 216, 129-139), etc. Hypoxanthine is onepreferred fully universal base. Nucleosides comprising hypoxanthineinclude, but are not limited to, inosine, isoinosine, 2′-deoxyinosine,and 7-deaza-2′-deoxyinosine, 2-aza-2′ deoxyinosine.

Additional universal bases are known in the art as described, forexample, in relevant portions of Loakes, D. and Brown, D. M., Nucl.Acids Res. 22:4039-4043, 1994; Ohtsuka, E. et al., J. Biol. Chem.260(5):2605-2608, 1985; Lin, P. K. T. and Brown, D. M., Nucleic AcidsRes. 20(19):5149-5152, 1992; Nichols, R. et al., Nature 369(6480):492-493, 1994; Rahmon, M. S, and Humayun, N. Z., Mutation Research 377(2): 263-8, 1997; Berger, M., et al., Nucleic Acids Research,28(15):2911-2914, 2000; Amosova, O., et al., Nucleic Acids Res. 25 (10):1930-1934, 1997; and Loakes, D., Nucleic Acids Res. 29(12):2437-47,2001. The universal base may, but need not, form hydrogen bonds with anoppositely located base. The universal base may form hydrogen bonds viaWatson-Crick or non-Watson-Crick interactions (e.g., Hoogsteeninteractions).

In certain embodiments of the invention rather than using anoligonucleotide probe comprising a universal base, an oligonucleotideprobe comprising an abasic residue is used. The abasic residue canoccupy a position opposite any of the four naturally occurringnucleotides and can thus serve the same function as a nucleotidecomprising a universal base. In some embodiments of the invention thelinkage adjacent to an abasic residue is cleaved by an AP endonuclease,but abasic residues are also of use as described here (i.e., to servethe function of a universal base) in embodiments in which other scissilelinkages (e.g., phosphorothiolates) are present and other cleavagereagents are used.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTIONA. Sequencing by Successive Cycles of Extension, Ligation, and Cleavage

The overall scheme of one aspect of the invention is showndiagrammatically in FIG. 1A, and generally resembles a method taught inU.S. Pat. Nos. 5,750,341 and 6,306,597, both issued to Macevicz. Forpurposes of convenience, these patents will be referred to collectivelyas “Macevicz” herein. In particular, Macevicz teaches a method foridentifying a sequence of nucleotides in a polynucleotide, the methodcomprising the steps of: (a) extending an initializing oligonucleotidealong the polynucleotide by ligating an oligonucleotide probe thereto toform an extended duplex; (b) identifying one or more nucleotides of thepolynucleotide; and (c) repeating steps (a) and (b) until the sequenceof nucleotides is determined.

Macevicz further teaches a method for determining a sequence ofnucleotides in a template polynucleotide, the method comprising thesteps of: (a) providing a probe-template duplex comprising aninitializing oligonucleotide probe hybridized to a templatepolynucleotide, said probe having an extendable probe terminus; (b)ligating an extension oligonucleotide probe to said extendable probeterminus, to form an extended duplex containing an extendedoligonucleotide probe; (c) identifying, in the extended duplex, at leastone nucleotide in the template polynucleotide that is either (1)complementary to the just-ligated extension probe or (2) a nucleotideresidue in the template polynucleotide which is immediately downstreamof the extended oligonucleotide probe; (d) generating an extendableprobe terminus on the extended probe, if an extendable probe terminus isnot already present, such that the terminus generated is different fromthe terminus to which the last extension probe was ligated; and (e)repeating steps (b), (c) and (d) until a sequence of nucleotides in thetarget polynucleotide is determined. In certain embodiments of thesemethods each extension probe has a chain-terminating moiety at aterminus distal to the initializing oligonucleotide probe. In certainembodiments the step of regenerating includes cleaving a chemicallyscissile internucleosidic linkage in the extended oligonucleotide probe.

Referring to FIG. 1A, polynucleotide template 20 comprising apolynucleotide region 50 of unknown sequence and binding region 40 isattached to support 10. Nucleotide 41, at the distal end of bindingregion 40, and nucleotide 51, at the proximal end of polynucleotideregion 50, are adjacent to one another. An initializing oligonucleotide30 is provided that hybridizes with binding region 40 to form a duplexat a location in binding region 40. Initializing oligonucleotide 30 isalso referred to as a “primer” herein, and binding region 40 may bereferred to as a “primer binding region”. The duplex may, but need notbe, a perfectly matched duplex. The initializing oligonucleotide has anextendable terminus 31. In FIG. 1A, the initializing oligonucleotidebinds to the binding region such that extendable terminus 31 is locatedopposite nucleotide 41. However, the initializing oligonucleotide couldbind elsewhere in the binding region, as discussed further below. Anextension oligonucleotide probe 60 of length N is hybridized to thetemplate adjacent to the initializing oligonucleotide. Terminalnucleotide 61 of the extension oligonucleotide probe is ligated toextendable terminus 31.

Terminal nucleotide 61 is complementary to the first unknown nucleotidein polynucleotide region 50. Therefore, the identity of terminalnucleotide 61 specifies the identity of nucleotide 51. Preferablynucleotide 51 is identified by detecting a label (not shown) associatedwith an extension probe known to have A, G, C, or T, as terminalnucleotide 61. The label is removed following detection. FIG. 2 shows ascheme for assigning different labels, e.g., fluorophores of differentcolors, to extension probes having different 3′ terminal nucleotides.

Following ligation and detection, an extendable probe terminus isgenerated on extension probe 60 if probe 60 does not already have such aterminus. A second extension probe 70, preferably also of length N, isannealed to the template adjacent to extension probe 60 and is ligatedto the extendable terminus of probe 60. The identity of terminalnucleotide 71 of extension probe 70 specifies the identity of oppositelylocated nucleotide 52 in polynucleotide 50. Terminal nucleotide 71therefore constitutes the “sequence determining portion” of theextension probe, by which is meant the portion of the probe whosehybridization specificity is used as a basis from which to determine theidentity of one or more nucleotides in the template. It will beappreciated that typically additional nucleotides in the extension probewill hybridize with the template, but only those nucleotides in theprobe whose identity is associated with a particular label are used toidentify nucleotides in the template.

In preferred embodiments of the invention, generation of the extendableterminus involves cleavage of an internucleoside linkage as describedfurther below. Preferably cleavage also removes the label. Cleavageremoves a number of nucleotides M from the extension probe (not shown).Therefore, the duplex is extended by N-M nucleotides in each cycle, andnucleotides located at intervals of N-M in the template are identified.It is to be understood that multiple copies of a given template willtypically be attached to a single support, and the sequencing reactionwill be performed simultaneously on these templates.

Macevicz teaches that the oligonucleotide probes should generally becapable of being ligated to an initializing oligonucleotide or extendedduplex to generate the extended duplex of the next extension cycle; theligation should be template-driven in that the probe should form aduplex with the template prior to ligation; the probe should possess ablocking moiety to prevent multiple probe ligations on the same templatein a single extension cycle; the probe should be capable of beingtreated or modified to regenerate an extendable end after ligation; andthe probe should possess a signaling moiety (i.e., a detectable moiety)that permits the acquisition of sequence information relating to thetemplate after a successful ligation.

Macevicz teaches characteristics of certain suitable initializingoligonucleotides, extension oligonucleotide probes, templates, bindingsites, and various methods for synthesizing, designing, producing, orobtaining such components. Macevicz further teaches certain suitableligases, ligation conditions, and a variety of suitable labels. Maceviczalso teaches an alternative method for identification using polymeraseextension to add a labeled chain-terminating nucleotide to a newlyligated extension probe. The identity of the added nucleotide identifiesthe nucleotide located oppositely in the template.

As will be appreciated by one of ordinary skill in the art, referencesto templates, initializing oligonucleotides, extension probes, primers,etc., generally mean populations or pools of nucleic acid molecules thatare substantially identical within a relevant region rather than singlemolecules. Thus, for example, a “template” generally means a pluralityof substantially identical template molecules; a “probe” generally meansa plurality of substantially identical probe molecules, etc. In the caseof probes that are degenerate at one or more positions, it will beappreciated that the sequence of the probe molecules that comprise aparticular probe will differ at the degenerate positions, i.e., thesequences of the probe molecules that constitute a particular probe maybe substantially identical only at the nondegenerate position(s). Forpurposes of description the singular form is to be understood to includesingle molecules and populations of substantially identical molecules.Where it is intended to refer to a single nucleic acid molecule (i.e.,one molecule), the terms “template molecule”, “probe molecule”, “primermolecule”, etc., will be used. In certain instances the plural nature ofa population of substantially identical nucleic acid molecules will beexplicitly indicated.

A population of substantially identical nucleic acid molecules may beobtained or produced using any of a variety of known methods includingchemical synthesis, biological synthesis in cells, enzymaticamplification in vitro from one or more starting nucleic acid molecules,etc. For example, using methods well known in the art, a nucleic acid ofinterest can be cloned by inserting it into a suitable expressionvector, e.g., a DNA or RNA plasmid, which is then introduced into cells,e.g., bacterial cells, in which it replicates. Plasmid DNA or RNAcontaining copies of the nucleic acid of interest is then isolated fromthe cells. Genomic DNA isolated from viruses, cells, etc., or cDNAproduced by reverse transcription of mRNA) can also be a source of apopulation of substantially identical nucleic acid molecules (e.g.,template polynucleotides whose sequence is to be determined) without anintermediate step of cloning or in vitro amplification, though generallyit is preferred to perform such an intermediate step.

It will be understood that members of a population need not be 100%identical, e.g., a certain number of “errors” may occur during thecourse of synthesis. Preferably at least 50% of the members of apopulation are at least 90%, or more preferably at least 95% identicalto a reference nucleic acid molecule (i.e., a molecule of definedsequence used as a basis for a sequence comparison). More preferably atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or more of the members of a population are at least 90%, ormore preferably at least 95% identical, or yet more preferably at least99% identical to the reference nucleic acid molecule. Preferably thepercent identity of at least 95% or more preferably at least 99% of themembers of the population to a reference nucleic acid molecule is atleast 98%, 99%, 99.9% or greater. Percent identity may be computed bycomparing two optimally aligned sequences, determining the number ofpositions at which the identical nucleic acid base (e.g., A, T, C, G, U,or I) occurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions, and multiplying the result by 100 to yield the percentage ofsequence identity. It will be appreciated that in certain instances anucleic acid molecule such as a template, probe, primer, etc., may be aportion of a larger nucleic acid molecule that also contains a portionthat does not serve a template, probe, or primer function. In that caseindividual members of a population need not be substantially identicalwith respect to that portion.

Macevicz teaches methods in which a template is attached to a supportsuch as a bead and extension proceeds towards the end of the templatethat is located distal to the support, as shown in FIG. 1A. Thus thebinding region is located closer to the support than the unknownsequence, and the extended duplex grows in the direction away from thesupport. However, the inventors have unexpectedly discovered that themethod can advantageously be practiced using an alternative approach inwhich the binding region is located at the end of the template that isdistal to the support, and extension proceeds inwards toward thesupport. This embodiment is depicted in FIG. 1B, in which the variouselements are numbered as in FIG. 1A. The inventors have determined thatsequencing “inwards” from the distal end of the template towards thesupport provides superior results. In particular, sequencing from thedistal end of the template towards a support such as a bead results inhigher ligation efficiencies than sequencing outwards from the support.

As further taught by Macevicz, preferably the oligonucleotide probes areapplied to templates as mixtures comprising oligonucleotides of allpossible sequences of a predetermined length. For example, a mixture ofprobes containing all possible sequences of 6 nucleotides in length(hexamers) of structure NNNNNN (which may also be represented as(N)_(k), where k=6) would contain 4⁶ (4096) probe species. Generally theprobes are of structure X(N)_(k)N*, where N represents any nucleotide,and k is between 1 and 100, * represents a label, and X represents anucleotide whose identity corresponds to the label. In certainembodiments k is between 1 and 100, between 1 and 50, between 1 and 30,between 1 and 20, e.g., between 4 and 10. One or more of the nucleotidesmay comprise a universal base. Generally the probe is 4-fold degenerateat positions represented by N or comprises a degeneracy-reducingnucleotide at one or more positions represented by N. If desired, themixture can be divided into subsets of probes (“stringency classes)whose perfectly matched duplexes with complementary sequences havesimilar stability or free energy of binding. The subsets may be used inseparate hybridization reactions as taught by Macevicz.

The complexity (i.e., the number of different sequences) of probemixtures can be reduced by a number of methods, including usingso-called degeneracy-reducing nucleotides or nucleotide analogs. Forexample, a library of probes containing all possible sequences of 8nucleotides would contain 4⁸ probes. The number of probes can be reducedto 4⁶ while retaining various desirable features of an octamer library,such as the length, by using universal bases at two of the positions.The present invention comprehends the use of any of the universal basesmentioned above or described in the references cited above.

Depending on the embodiment, the extended duplex or initializingoligonucleotide may be extended in either the 5′→3′ direction or the3′→5′ direction by oligonucleotide probes, as described further below.Generally, the oligonucleotide probe need not form a perfectly matchedduplex with the template, although such binding may be preferred. Inembodiments in which a single nucleotide in the template is identifiedin each extension cycle, perfect base pairing is only required foridentifying that particular nucleotide. For example, in embodimentswhere the oligonucleotide probe is enzymatically ligated to an extendedduplex, perfect base pairing, i.e. proper Watson-Crick base pairing, isrequired between the terminal nucleotide of the probe which is ligatedand its complement in the template. Generally, in such embodiments, therest of the nucleotides of the probe serve as “spacers” that ensure thenext ligation will take place at a predetermined site, or number ofbases, along the template. That is, their pairing, or lack thereof, doesnot provide further sequence information. Likewise, in embodiments thatrely on polymerase extension for base identification, the probeprimarily serves as a spacer, so specific hybridization to the templateis not critical.

The methods described above allow partial determination of a sequence,i.e., the identification of individual nucleotides spaced apart from oneanother in a template. In preferred embodiments of the invention, inorder to gather more complete information, a plurality of reactions isperformed in which each reaction utilizes a different initializingoligonucleotide i. The initializing oligonucleotides i bind to differentportions of the binding region. Preferably the initializingoligonucleotides bind at positions such the extendable termini of thedifferent initializing oligonucleotides are offset by 1 nucleotide fromeach other when hybridized to the binding region. For example, as shownin FIG. 3, sequencing reactions 1 . . . N are performed. Initializingoligonucleotides i₁ . . . i_(n) have the same length and bind such thattheir terminal nucleotides 31, 32, 33, etc., hybridize to successiveadjacent positions 41, 42, 43, etc., in binding region 40. Extensionprobes e₁ . . . e_(n) thus bind at successive adjacent regions of thetemplate and are ligated to the extendable termini of the initializingoligonucleotides. Terminal nucleotide 61 of probe e_(n) ligated to i_(n)is complementary to nucleotide 55 of polynucleotide region 50, i.e., thefirst unknown polynucleotide in the template. In the second cycle ofextension, ligation, and detection, terminal nucleotide 71 of probe e₁₂is complementary to nucleotide 56 of polynucleotide region 50, i.e., thesecond nucleotide of unknown sequence. Likewise, terminal nucleotides ofextension probes ligated to duplexes initialized with initializingoligonucleotides i₂, i₃, i₄, and so on, will be complementary to thethird, fourth, and fifth nucleotides of unknown sequence 50. It will beappreciated that the initializing oligonucleotides may bind to regionsprogressively further away from polynucleotide region 50 rather thanprogressively closer to it.

The spacer function of the non-terminal nucleotides of the extensionprobes allows the acquisition of sequence information at positions inthe template that are considerably removed from the position at whichthe initializing oligonucleotide binds without requiring acorrespondingly large number of cycles to be performed on any giventemplate. For example, by successive cycles of ligation of probes oflength N, followed by cleavage to remove a single terminal nucleotidefrom the extension probe, nucleotides at intervals of N-1 nucleotidescan be identified in successive rounds. For example, nucleotides atpositions 1, N, 2N-1, 3N-2, 4N-3, and 5N-4 in the template can beidentified in 6 cycles where the nucleotide at position 1 in thetemplate is the nucleotide opposite the nucleotide that is ligated tothe extendable probe terminus in the duplex formed by the binding of theinitializing oligonucleotide to the template. Similarly, if cleavageremoves two nucleotides from the extension probes of length N, thennucleotides at positions separated from each other by N-2 nucleotidescan be identified in successive rounds. For example, nucleotides atpositions 1, N-1, 2N-3, 3N-5, 4N-7 in the template can be identified in6 cycles. Thus if the probes are 8 nucleotides in length and 2nucleotides are removed in each cycle, nucleotides at positions 1, 7,13, 19, and 25 are identified. Thus the number of cycles needed toidentify a nucleotide at a distance X from the first nucleotide in thetemplate is on the order of X/M, where M is the length of the extensionprobe that remains following cleavage, rather than on the order of X.

For example, the schematic depicted in FIG. 3B shows the net result ofusing the extension, ligation, and cleavage method with extension probesdesigned to read every 6th base of the template. By serially strippingand sequencing the template using 6 initializing nucleotides that bindto positions that are offset within the binding region and combining theresults, all template bases are elucidated over a defined length. Forinstance, if 10 serial ligations are performed for each of the 6reactions, the resulting read length will be 60 sequential base pairs,whereas if 15 serial ligations are performed for each reaction theresultant read length will be 90 sequential base pairs.

While not wishing to be bound by any theory, the inventors suggest thatin contrast to this approach, most serial sequencing by synthesismethods struggle with error accumulation that ultimately limits thepotential for long read lengths. An advantageous feature of certain ofthe methods described herein is that they allow the identification ofevery n^(th) base (depending on the position of the cleavable moiety inthe probe), such that after a given number of cycles (y), one reachesthe n*y−(n−1)^(th) base (e.g., the 71^(st) base in the foregoing exampleafter 15 cycles, or the 115^(th) base after 20 cycles using a probe with6 bases on the 3′ side of the cleavage site). The ability to “reset” theinitializing oligonucleotide at the n-1, n-2, etc., positions greatlyminimizes serial error accumulation (via dephasing or attrition) for agiven read length since the process of stripping the extended strandsfrom the template and hybridizing a new initializing oligonucleotideeffectively resets background signals to zero. For example, comparingthe polymerase based sequencing by synthesis and the ligation basedapproaches described herein, if the signal to noise ratio at eachextension cycle is 99:1, the ratio after 100 cycles for the polymerasebased approach will be 37:63 and for the ligase based method, 85:15. Thenet result for the ligase based method is a large increase in readlength over polymerase based methods.

The ability to identify nucleotides using fewer cycles than would berequired if it was necessary to perform a cycle for each precedingnucleotide in the template is important for a number of reasons. Inparticular, it is unlikely that each step in the method will occur with100% efficiency. For example, some templates may not be successfullyligated to an extension probe; some extension probes may not be cleaved,etc. Thus in each cycle the reactions occurring on different copies ofthe template become progressively dephased, and the number of templatesfrom which useful and accurate information can be acquired is reduced.It is thus particularly desirable to minimize the number of cyclesrequired to read nucleotides located more than a few positions away fromthe extendable terminus of the initializing oligonucleotide. However,increasing the length of the extension probe potentially results ingreater complexity of the probe mixture, which decreases the effectiveconcentration of each individual probe sequence. As described herein,degeneracy-reducing nucleotides can be used to reduce the complexity butmay result in decreased hybridization strength and/or decreased ligationefficiency. The inventors have recognized the need to balance thesecompeting factors in order to optimize results. Thus in a preferredembodiment of the invention extension probes 8 nucleotides in length areused, with degeneracy-reducing nucleotides at selected positions. Inaddition, the inventors have recognized the importance of selectingappropriate scissile linkages and cleavage conditions and times tooptimize the efficiency of the cleavage step (i.e., the percentage oflinkages that is successfully cleaved in each cleavage step) and itsspecificity for the appropriate linkage.

B. Oligonucleotide Extension Probe Design

While Macevicz mentions that degeneracy-reducing nucleoside analogs maybe used in the oligonucleotide extension probes, he does not teachspecific positions at which it is particularly desirable to include aresidue comprising such residues in the extension probes and does notteach particular probe structures (i.e., sequences) that incorporatedegeneracy-reducing nucleosides. The present inventors have recognizedthat it may be particularly advantageous to utilize degeneracy-reducingnucleosides (e.g., nucleosides that comprise a universal base) atparticular positions and in particular numbers in the oligonucleotideextension probes. For example, in certain embodiments of the inventionmost or all of the nucleotides at position 6 or greater (counting fromX), comprise a universal base. For example, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, or at least 100% of thenucleotides at position 6 or greater may comprise a universal base. Thenucleotides need not all comprise the same universal base. In certainembodiments of the invention hypoxanthine and/or a nitro-indole is usedas a universal base. For example, nucleosides such as inosine can beused.

The inventors have recognized that superior results may be achievedusing extension probes that are greater than 6 nucleotides in length,and in which one or more of the nucleotides at position 6 or greaterfrom the proximal terminus of the probe, counting from the nucleotide tobe ligated to the extendable probe terminus, is a degeneracy-reducingnucleotide, e.g., comprises a universal base (i.e., if the most proximalnucleotide is considered position 1, one or more of the nucleotides atposition 6 or greater comprises a universal base), e.g., 1, 2, or 3 ofthe nucleotides at position 6 or greater in the case of octamer probescomprises a universal base. For example, for sequencing in the 3′→5′direction, probes having the structure 3′-XNNNNsINI-5′ can be used,where X and N represent any nucleotide, “s” represents a scissilelinkage, such that cleavage occurs between the fifth and sixth residuescounting from the 3′ end, and at least one of the residues between thescissile linkage and the 5′ end preferably has a label that correspondsto the identity of X. Another design is 3′-XNNNNsNII-5′. Yet anotherprobe design is 3′-XNNNNsIII-5′. This design yields a probe mixture witha modest complexity of 1024 different species, is long enough to preventformation of significant adenylation products (see Example 1), and hasthe advantage that the resulting extension product remaining aftercleavage would consist of unmodified DNA. One drawback is that thisprobe extends the primer by only 5 bases at a time. Since the readlength is a function of the extension length times the number of cycles,each additional base on the extension length has the potential toincrease the read length by the 1× the cycle number (e.g. 20 bases if 20cycles are used). Another probe design leaves one or more inosines (orother universal base) at the end of the extension probe followingcleavage to create a 6 base, or longer, extended duplex. For example,with the probe 3′-XNNNNIsII-5′, the duplex would be extended by 6 basesat a time, leaving a 5′ inosine at the junction. In each of thesedesigns, at least one of the residues between the scissile linkage andthe 5′ end preferably has a label that corresponds to the identity of X.In certain embodiments of the invention the third nucleotide from thedistal terminus of the probe, counting from the end opposite thenucleotide to be ligated to the extendable probe terminus, comprises auniversal base, (i.e., if the distal terminus is considered position K,the nucleotide at position K-2 comprises a universal base).

In certain embodiments of the invention locked nucleic acid (LNA) basesare used at one or more positions in an initializing oligonucleotideprobe, extension probe, or both. Locked nucleic acids are described, forexample, in U.S. Pat. No. 6,268,490; Koshkin, A A, et al., Tetrahedron,54:3607-3630, 1998; Singh, S K, et al., Chem. Comm., 4:455-456, 1998.LNA can be synthesized by automatic DNA synthesizers using standardphosphoramidite chemistry and can be incorporated into oligonucleotidesthat also contain naturally occurring nucleotides and/or nucleotideanalogues. They can also be synthesized with labels such as thosedescribed below.

C. Template and Support Preparation Methods

Macevicz teaches a process in which a template comprising a plurality ofsubstantially identical template molecules is first synthesized, e.g.,by amplification in a tube or other vessel as in conventional polymerasechain reaction (PCR) methods. Macevicz teaches that the amplifiedtemplate molecules are preferably attached to supports such as magneticmicroparticles (e.g., beads) after synthesis.

The inventors have recognized that templates to be sequenced maydesirably be synthesized on or in a support itself, e.g., by usingsupports such as microparticles or various semi-solid support materialssuch as gel matrices to which one of a pair of amplification primers isattached prior to performing the PCR reaction. This approach avoids theneed for a separate step of attaching the template molecules to thesupport after synthesis. Thus a plurality of template species ofdiffering sequence can be conveniently amplified in parallel. Forexample, according to the methods described below, synthesis onmicroparticles results in a population of individual microparticles,each with multiple copies of a particular template molecule (or itscomplement) attached thereto, wherein the template molecules attached toeach microparticle differ in sequence from the template moleculesattached to other microparticles. Each of the supports thus has a clonalpopulation of templates attached thereto, e.g., support A will havemultiple copies of template X attached thereto; support B will havemultiple copies of template Y attached thereto; support C will havemultiple copies of template Z attached thereto, etc. By “clonalpopulation of templates”, “clonal population of nucleic acids”, etc., ismeant a population of substantially identical template molecules,preferably generated by successive rounds of amplification that startfrom a single template molecule of interest (starting template). Thesubstantially identical template molecules may be substantiallyidentical to the starting template or to its complement.

Amplification is typically performed using PCR, but other amplificationmethods may also be used (see below). It will be understood that membersof a clonal population need not be 100% identical, e.g., a certainnumber of “errors” may occur during the course of synthesis, e.g.,during amplification. Preferably at least 50% of the members of a clonalpopulation are at least 90%, or more preferably at least 95% identicalto a starting template molecule (or to its complement). More preferablyat least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or more of the members of a population are at least 90%, ormore preferably at least 95% identical, or yet more preferably at least99% identical to the starting template molecule (or to its complement).Preferably the percent identity of at least 95% or more preferably atleast 99% of the members of the population to a starting templatemolecule (or to its complement) is at least 98%, 99%, 99.9% or greater.

Amplification primers may be attached to supports using any of a varietyof techniques. For example, one end of the primer (the 5′ end) of theprimer may be functionalized with one member of a binding pair (e.g.,biotin), and the support functionalized with the other member of thebinding pair (e.g., streptavidin). Any similar binding pair may be used.For example, nucleic acid tags of defined sequence may be attached tothe support and primers having complementary nucleic acid tags can behybridized to the nucleic acid tags attached to the support. Variouslinkers and crosslinkers can also be used.

Methods for performing PCR are well known in the art and are described,for example, in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, andin Dieffenbach, C. and Dveksler, G S, PCR Primer: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,2003. Methods for amplifying nucleic acids on microparticles are wellknown in the art and are described, for example, standard PCR can beperformed in wells of a microtiter dish or in tubes on beads withprimers attached thereto (e.g., beads prepared as in Example 12. WhilePCR is a convenient amplification method, any of numerous other methodsknown in the art can also be used. For example, multiple stranddisplacement amplification, helicase displacement amplification (HDA),nick translation, Q beta replicase amplification, rolling circleamplification, and other isothermal amplification methods etc., can beused.

Template molecules can be obtained from any of a variety of sources. Forexample, DNA may be isolated from a sample, which may be obtained orderived from a subject. The word “sample” is used in a broad sense todenote any source of a template on which sequence determination is to beperformed. The phrase “derived from” is used to indicate that a sampleand/or nucleic acids in a sample obtained directly from a subject may befurther processed to obtain template molecules. The source of a samplemay be of any viral, prokaryotic, archaebacterial, or eukaryoticspecies. In certain embodiments of the invention the source is a human.The sample may be, e.g., blood or another body fluid containing cells;sperm; a biopsy sample, etc. Genomic or mitochondrial DNA from anyorganism of interest may be sequenced. cDNA may be sequenced. RNA mayalso be sequenced, e.g., by first reverse transcribing to yield cDNA,using methods known in the art such as RT-PCR. Mixtures of DNA fromdifferent samples and/or subjects may be combined. Samples may beprocessed in any of a variety of ways. Nucleic acids may be isolated,purified, and/or amplified from a sample using known methods. Of courseentirely artificial, synthetic nucleic acids, recombinant nucleic acidsnot derived from an organism can also be sequenced.

Templates can be provided in double or single-stranded form. Typicallywhen a template is initially provided in double-stranded form the twostrands will subsequently be separated (e.g., the DNA will bedenatured), and only one of the two strands will be amplified to producea localized clonal population of template molecules, e.g., attached to amicroparticle, immobilized in or on a semi-solid support, etc.

Templates may be selected or processed in a variety of additional ways.For example, templates obtained from DNA that has been subjected totreatment to with a methyl-sensitive restriction enzyme (e.g., MspI) canbe used. Such treatment, which results in DNA fragments, can beperformed prior to amplification. Fragments containing methylated basesdo not amplify. Sequence information obtained from the hypomethylatedtemplates may be compared with sequence information obtained fromtemplates derived from the same source, which were not subjected toselection for hypomethylation.

Templates may be inserted into, provided in, or derived from a library.For example, hypomethylated libraries are known in the art. Insertingtemplates into libraries can allow for the convenient concatenation ofadditional nucleotide sequences to the ends of templates, e.g., tags,binding sites for primers or initializing oligonucleotides, etc. Forexample, certain strategies allow the addition of tags having aplurality of binding sites, e.g., a binding site for an amplificationprimer, a binding site for an initializing oligonucleotide, a bindingsite for a capture agent, etc.

A variety of suitable libraries are known in the art. For example,libraries of particular interest, and methods for their construction,are described in U.S. Ser. No. 10/978,224, PCT publications WO2005042781and WO2005082098, and Shendure, J., et al., Science, 309(5741):1728-32,2005, Sciencexpress, 4 Aug. 2005 (www.sciencexpress.org). Of course itwill be understood that other methods of generating such libraries couldalso be used. Certain libraries of particular interest contain aplurality of nucleic acid fragments (typically DNA), each of whichcontain two nucleic acid segments of interest, separated by sequencesthat are complementary to amplification and/or sequencing primers thatare used in sequencing steps, i.e., these sequences serve as primerbinding regions (PBRs). In embodiments of particular interest, thenucleic acid segments are portions of a contiguous piece of naturallyoccurring DNA. For example, the segments may be from the 5′ and 3′ endof a contiguous piece of genomic DNA as described in the afore-mentionedreferences. Such nucleic acid segments are referred to herein in amanner consistent with the afore-mentioned references, as “tags” or “endtags”. Two tags derived from a single contiguous nucleic acid, e.g.,from the 5′ and 3′ ends thereof, are referred to as “a paired tag”,“paired tags”, or “a ditag”. It will be appreciated that a “paired tag”comprises two tags, even if used in the singular. By selecting thecontiguous pieces of DNA from which the tags of a paired tag are derivedto be within a predefined size limit, the distance separating the twotags is constrained.

In addition to being separated by sequences that are complementary tosequencing and/or amplification primers, the nucleic acid fragments ofthe libraries typically also contain sequences complementary tosequencing and/or amplification primers flanking the tags, i.e., a firstsuch sequence may be located 5′ to the tag that is closer to the 5′ endof the fragment, and a second such sequence may be located 3′ to the tagthat is located closer to the 3′ end of the fragment. It is noted thatthe position of the two tags as present in the contiguous nucleic acidfrom which the tags are derived may, but need not, correspond with theposition of the tag in the DNA fragment of the library in variousembodiments.

The nucleic acid fragments and the tags can have a range of differentsizes. Typically the nucleic acid fragments may be, for example, between80 and 300 nucleotides in length, e.g., between 100-200, 100-150,approximately 150 nucleotides in length, approximately 200 nucleotidesin length, etc. The tags can be, e.g., between 15-25 nucleotides inlength, e.g., approximately 17-18 nucleotides in length, etc. It isnoted that these lengths are exemplary and are not intended to belimiting. Shorter or longer fragments and/or tags could be used.

It should also be noted that while obtaining the paired tags from asingle contiguous nucleic acid affords a convenient method for libraryconstruction, the important aspect of the paired tags is the fact thatthey are separated from one another by a distance (“separationdistance”) in the nucleic acid from which they were originally derived,wherein the separation distance falls within a predetermined range ofdistances. The fact that the tags are separated by a separation distancethat falls within a predetermined range allows the sequence of the tagsto be aligned against a reference sequence (e.g., a reference genomesequence). Without wishing to be bound by any theory, this can beadvantageous in certain applications such as genome resequencing,wherein it allows the use of shorter read lengths while still allowingaccurate placement of the sequences with respect to the referencegenome. The 5′ and 3′ tags of a paired tag represent (i.e., they havethe sequence of) segments of a larger piece of nucleic acid, e.g.,genomic DNA, which segments are located within a predefined distancefrom one another in a naturally occurring piece of DNA, e.g., within apiece of genomic DNA. For example, in certain embodiments of theinvention the 5′ and 3′ tags of a paired tag represent segments of DNAlocated within up to 500 nucleotides of each other, within up to 1 kB ofeach other, within up to 2 kB of each other, within up to 5 kB of eachother, within up to 10 kB of each other, within up to 20 kB of eachother, in a naturally occurring piece of DNA. In certain embodiments the5′ and 3′ tags of a paired tag are located between 500 nucleotides and 2kB apart, e.g, between 700 nucleotides and 1.2 kB apart, approximately 1kB apart, etc., in a naturally occurring piece of DNA. It is noted thatthe exact distance separating the two tags of a paired tag is not ofmajor importance and is typically not known. In addition, while the tagsare originally obtained from a larger piece of nucleic acid, the word“tag” applies to any nucleic acid segment that has the sequence of thetag, whether present in its original sequence context or in a libraryfragment, amplification product from a library fragment, template to besequenced, etc.

A nucleic acid fragment (e.g., a library molecule) may have thefollowing structure:

Linker 1-Tag 1-Linker 3-Tag 1-Linker 2

Tag 1 and Tag 2 can be 5′ and 3′ tags of a paired tag. Either of thetags can be the 5′ tag or the 3′ tag. Linker 1 and Linker 2 containprimer binding regions for one or more primers. In certain embodimentsLinkers 1 and 2 each contain a PBR for an amplification primer and a PBRfor a sequencing primer. The primers in each linker can be nested, suchthat the sequencing primer PBR is located internal to the amplificationprimer PBR. Linker 3 may contain PBRs for one or more sequencing primersto allow for sequencing of Tag 1 and Tag 2. The term “linker” refers toa nucleic acid sequence that is present in multiple nucleic acidfragments of a library, e.g., in substantially all fragments of thelibrary. A linker may or may not actually have served a linking functionduring construction of the library and can simply be considered to be adefined sequence that is common to most or all members of a givenlibrary. Such a sequence is also referred to as a “universal sequence”.Thus a nucleic acid complementary to the linker or a portion thereofwould hybridize to multiple members of the library and could be used asan amplification primer or sequencing primer for most or all moleculesin the library.

In certain embodiments of the present invention, a nucleic acid fragmenthas the following structure:

Linker 1-Tag 1-Internal Adaptor-Tag 2-Linker 2

Tag 1 and Tag 2 and Linker 1 and Linker 2 contain PBRs as describedabove. Internal Adaptor contains two primer binding regions, which maybe referred to as IA and IB, as discussed further below. These PBRs areof use to produce microparticles having two distinct substantiallyidentical populations of nucleic acids attached thereto, wherein nucleicacids of one of the populations comprise Tag 1 and nucleic acids of theother population comprise Tag 2. The two distinct populations of nucleicacids have at least partially different sequences, e.g., they differ inthe sequence of the tag regions. The Internal adaptor can contain aspacer region between the two primer binding regions. The spacer regionmay contain abasic residues, which will prevent a polymerase fromextending through the spacer. Of course spacer regions containing anyother blocking group that would prevent polymerase extension through thespacer could be used.

In other embodiments, a nucleic acid fragment includes one or moreadditional tags (e.g, 2, 4, 6, etc.) and one or more additional internaladaptors. For example, a nucleic acid fragment can have the followingstructure:

Linker 1-Tag 1-Internal Adaptor 1-Tag 2-Linker 2-Tag 3-Internal Adaptor2-Tag 4-Linker 3

It is noted that the inventive nucleic acid fragments and libraries ofsuch fragments, microparticles containing two or more substantiallyidentical populations of nucleic acids, and arrays of suchmicroparticles can be used in a wide variety of sequencing methods otherthan the ligation-based sequencing methods described herein. Forexample, sequencing methods such as FISSEQ, pyrosequencing, etc., can beused. See, e.g., WO2005082098. Of course the ligation-based methods canalso advantageously be employed. It will be appreciated that in thecontext of the ligation-based methods described herein, the term“sequencing primer” may be understood to mean “initializingoligonucleotide”.

In certain embodiments of the invention the templates to be sequencedare synthesized by PCR in individual aqueous compartments (also called“reactors”) of an emulsion. Preferably the compartments each contain aparticulate support such as a bead having a suitable first amplificationprimer attached thereto, a first copy of the template, a secondamplification primer, and components needed for the PCR reaction (e.g.,nucleotides, polymerase, cofactors, etc.). Methods for preparingemulsions are described for example, in U.S. Pat. No. 6,489,103(Griffiths); U.S. Pat. No. 5,830,663 (Embleton); and in U.S. Pub. No.20040253731 (Ghadessy). Methods for performing PCR within individualcompartments of an emulsion to produce clonal populations of templatesattached to microparticles are described, e.g., in Dressman, D., et al.,Proc. Natl. Acad. Sci., 100(15):8817-8822, 2003, and in PCT publicationWO2005010145.

Methods described in the afore-mentioned references, or modificationsthereof, may be used to produce clonal populations of templates attachedto microparticles for sequencing. In a preferred and non-limitingembodiment, short (<500 nucleotide) templates suitable for PCR arecreated by attaching (e.g., by ligation) a universal adaptor sequence toeach end of a population of different target sequences (templates).(Universal in this context means that the same adaptor sequence isattached to each template, to create “adapted” templates that can beamplified using a single pair of PCR amplification primers.) A bulk PCRreaction is prepared with the adapted templates, one free amplificationprimer, microparticles with a second amplification primer attachedthereto, and other PCR reagents (e.g., polymerase, cofactors,nucleotides, etc.). The aqueous PCR reaction is mixed with an oil phase(containing light mineral oil and surfactants) in a 1:2 ratio. Thismixture is vortexed to create a water-in-oil emulsion. One milliliter ofmixture is sufficient to create more than 4×10⁹ aqueous compartmentswithin the emulsion, each a potential PCR reactor. Aliquots of theemulsion sample are dispensed into the wells of a microtiter plate(e.g., 96 well plate, 384 well plate, etc.) and thermally cycled toachieve solid-phase PCR amplification on the microparticles. To ensureclonality, the microparticle and template concentrations are carefullycontrolled so that the reactors rarely contain more than one bead ortemplate molecule. For example, in certain embodiments of the inventionat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more ofthe reactors contain a single bead and a single template. Members ofeach clonal populations of templates are thus spatially localized inproximity to one another as a result of their attachement to themicroparticle. In general, the points of attachment of the templates maybe substantially uniformly distributed on the surface of the particle.

It is of particular interest to use PCR emulsion methods to producepopulations of microparticles in which individual microparticles havedistinct populations of amplified nucleic acid fragments that contain a5′ tag and a 3′ tag of a paired tag attached thereto. In other words, itis of particular interest to produce populations of microparticles inwhich individual particles have different nucleic acid fragments from alibrary such as those described above amplified and attached thereto.

Methods known in the art for amplifying DNA in emulsions (e.g.,described in the references mentioned above), are limited in terms oftheir ability to achieve amplification of large nucleic acid moleculesand attachment of these molecules to microparticles. For example, it hasbeen demonstrated that the PCR efficiency decays exponentially withlonger amplicons. This decrease in PCR efficiency reduces the efficiencywith which nucleic acid fragments containing paired tags and primerbinding sites, such as those described above, can be amplified in PCRemulsions and attached to microparticles via such amplification. Thusmethods in which a single population of substantially identical nucleicacid fragments containing first and second tags of a paired tag areamplified in a PCR emulsion and attached to beads via such amplificationsuffer from a number of limitations.

The present invention provides an approach that allows the use ofsmaller amplicons while still preserving the paired tag information thatarises when a single nucleic acid fragment containing 5′ and 3′ tags ofa paired tags is attached via amplification to a microparticle. Theinvention provides a microparticle, e.g., a bead, having at least twodistinct populations of nucleic acids attached thereto, wherein each ofthe at least two populations consists of a plurality of substantiallyidentical nucleic acids, and wherein a first population of substantiallyidentical nucleic acids comprises a first nucleic acid segment ofinterest, e.g., 5′ tag, and a second population of nucleic acidscomprises a second nucleic acid segment of interest, e.g., 3′ tag. Thefirst and second populations of nucleic acids are amplified from asingle larger nucleic acid fragment that contains the two tags and alsocontains appropriately positioned primer binding sites flanking andseparating the tags, so that two amplification reactions can beperformed either sequentially or, preferably, simultaneously, in asingle reactor of a PCR emulsion in the presence of a microparticle andamplification reagents. The microparticle has attached thereto twodifferent populations of primers, one of which corresponds in sequencewith a primer binding region external to one of the tags in the nucleicacid fragment, and the other of which corresponds in sequence with aprimer binding region external to the other tag in the nucleic acidfragment, i.e., the primer binding regions flank the two tags.

Also provided are primers that bind to primer binding regions locatedbetween the two tags, so that two separate PCR reactions can beperformed, each amplifying a portion of the nucleic acid fragmentcontaining one of the tags. The amplified nucleic acid segments containadditional primer binding regions, which are different from one another.These additional primer binding regions are present in the nucleic acidfragment and are located internal to the PBRs for the amplificationprimers, i.e., they are nested. These additional PBRs serve as bindingregions for two different sequencing primers. Thus by applying one orthe other of the two different sequencing primers to a microparticlehaving the two populations of substantially identical nucleic acidsegments attached thereto, either one or the other of the two nucleicacid segments can be sequenced without interference due to the presenceof the other nucleic acid segment. Each of the nucleic acid segments issignificantly shorter than the nucleic acid fragment from which it wasamplified, thus improving the efficiency with which emulsion-based PCRcan be performed using libraries of fragments containing paired tags,while still preserving the association between the tags of a paired tag.

The methods described above may be better understood by reference to thevarious panels of FIGS. 34 and 35 in which portions of nucleic acidshaving the same sequence are assigned the same color. The descriptionabove is to be interpreted consistently with FIGS. 34 and 35. FIGS. 34Aand 35A show the same steps, with FIG. 35A providing additional details.As shown in FIGS. 34A and 35A, paired-end library fragments containingtwo tags (Tag 1 and Tag 2) are constructed with an internal adaptercassette (IA-IB) and unique flanking linker sequences (P1 and P2). Boththe internal adapter cassette and the flanking linker sequences containnucleotide sequences that afford both PCR amplification and DNAsequencing. PCR primer regions are designed as to allow the use ofnested DNA sequencing primers. DNA capture microparticles (beads) aregenerated by attaching two oligonucleotide sequences that are identicalto the unique flanking linker sequences. For PCR amplification, DNAcapture microparticles bound with oligonucleotides having P1 and P2sequences, are seeded into reactions containing a single di-tag libraryfragment (i.e., a library fragment containing a 5′ tag and 3′ tag of apaired tag) and solution-based PCR primers.

Solution-based flanking linker primers (P1 and P2) are added in limitingamounts in comparison to the internal adapter primers (IA and IB) andwill serve to promote efficient drive-to-bead amplification ofPCR-generated tag products (i.e., [P1<<IB], [P2<<IA]). If desired,controlling the amount of primers appropriately can also ensure that thepopulations of nucleic acids contain substantially the same number ofnucleic acids, e.g., approximately half the nucleic acids on anindividual microparticle belong to the first population andapproximately half the nucleic acids on an individual microparticlebelong to the second population. Thus a form of asymmetric PCR can beemployed, if desired, in order to control the ratio of the differentpopulations.

During amplification, as shown in FIGS. 34B and 35B (where FIG. 35Bagain provides additional details relative to FIG. 34B), the singlepaired-end library fragment, in the presence of the four oligonucleotideprimers (P1, P2, IA and IB), will generate two unique PCR products. Onepopulation contains Tag 1 flanked by P1 and IA, and a second populationcontains Tag 2 flanked by P2 and IB.

Following amplification microparticles will be loaded with two uniquePCR populations corresponding to Tag 1 and Tag 2 generated from theinitial library fragment. Each tag thus contains a unique set of primingregions to allow serial sequencing of each tag as shown in FIGS. 34C,35C, and 35D. FIGS. 35C and 35D show sequential sequencing of tags 1 and2, using different sequencing primers. Any of a variety of sequencingmethods can be used.

The above methods can be used to generate microparticles having morethan two distinct populations of nucleic acid sequences attachedthereto, e.g., 4, 6, 8, 12, 16, 20, populations, e.g., wherein thepopulations comprise 2, 3, 4, 6, 8, 10 paired tags. Each population canbe individually sequenced by providing a unique primer binding region ineach sequence, as described above in the case of two tags.

The invention encompasses nucleic acid fragments having the structuresshown in FIGS. 34 and 35 and described above, libraries of suchfragments, microparticles having nucleic acid segments from suchfragments attached thereto, populations of such microparticles whereinthe individual microparticles have populations of nucleic acids attachedthereto that differ in sequence from those of other microparticles,arrays of microparticles, amplification primers for amplifying nucleicacid segments (tags) from the nucleic acid fragments, sequencing primersfor sequencing nucleic acid segments attached to microparticles, methodsfor making the fragments, libraries and microparticles, and methods ofsequencing the nucleic acids attached to the microparticles. Theinvention encompasses kits containing any combination of theafore-mentioned components, optionally also containing one or moreenzymes, buffers, or other reagents useful in amplification, sequencing,etc.

If desired, a variety of methods may be used to enrich formicroparticles that have templates attached thereto. For example, ahybridization-based method can be used in which an oligonucleotide(capture agent) complementary to a portion of an amplification product(template) attached to the microparticles is attached to a captureentity such as another (preferably larger) microparticle, microtiterwell, or other surface. The portion of the amplification product may bereferred to as a target region. The target region may be incorporatedinto templates during amplification, e.g., at one end of the portion ofthe template having unknown sequence. For example, the target region maybe present in the amplification primers that is not attached to themicroparticle, so that a complementary portion is present in theamplified template. Thus multiple different templates can include thesame target region, so that a single capture agent will hybridize tomultiple different templates, allowing the capture of multiplemicroparticles using only a single oligonucleotide sequence as thecapture agent. Microparticles that have been subjected to amplificationare exposed to the capture agent under conditions in which hybridizationcan occur. As a result, microparticles having amplified templatesattached thereto are attached to the capture entity via the captureagent. Unattached microparticles are then removed, and the retainedmicroparticles released (e.g., by raising the temperature). In certainembodiments in which a particulate capture entity is used, aggregatesconsisting of the capture entity with microparticles attached theretoafter hybridization are separated from particulate capture entitieslacking attached microparticles and from microparticles that are notattached to a capture entity, e.g., by centrifugation in a viscoussolution such as glycerol. Other methods of separation based on size,density, etc., can also be used. Hybridization is but one of a number ofmethods that can be used for enrichment. For example, capture agentshaving an affinity for any of a number of different ligands that can beincorporated into a template (e.g., during synthesis) may be used.Multiple rounds of enrichment can be used.

FIG. 14A shows an image of compartments of a water-in-oil emulsion, inwhich PCR reactions were performed on beads having first amplificationprimers attached thereto, using a fluorescently labeled secondamplification primer and an excess of template. Aqueous reactorsfluoresce weakly from diffuse free primer whereas beads stronglyfluoresce from primers accumulating on the bead as a result ofsolid-phase amplification (i.e., fluorescent primers are incorporatedinto the amplified templates that are attached to the beads via thefirst amplification primer). Bead signal is uniform in the differentsized reactors.

Following amplification, microparticles are collected (e.g., by use of amagnet in the case of magnetic particles) and used for sequencing byrepeated cycles of extension, ligation, and cleavage as describedherein. In certain embodiments of the invention the microparticles arearrayed in or on a semi-solid support prior to sequencing, as describedbelow. Examples 12, 13, 14, and 15 provide additional details ofrepresentative and nonlimiting methods that may be used to (i) preparemicroparticles having an amplification primer attached thereto, forsynthesis of templates on the microparticles (Example 12); (ii)preparation of an emulsion comprising a plurality of reactors forperforming PCR (Example 13); (iii) PCR amplification in compartments ofan emulsion (Example 13); (iv) breaking the emulsion and recoveringmicroparticles (Example 13); (v) enriching for microparticles havingclonal template populations attached thereto (Example 14); (vi)preparation of glass slides to serve as substrates for a semi-solidpolyacrylamide support (Example 15); and (vii) mixing microparticleswith unpolymerized acrylamide, forming an array of microparticles havingtemplates attached thereto, embedded in acrylamide on a substrate(Example 15). Example 15 also describes a protocol for polymerasetrapping, which is used in certain of the methods when performing PCR ina semi-solid support. One of ordinary skill in the art will recognizethat numerous variations on these methods may be used.

In other embodiments of the invention, the templates are amplified byPCR in a semi-solid support such as a gel having suitable amplificationprimers immobilized therein. Templates, additional amplificationprimers, and reagents needed for the PCR reaction are present within thesemi-solid support. One or both of a pair of amplification primers isattached to the semi-solid support via a suitable linking moiety, e.g.,an acrydite group. Attachment may occur during polymerization.Additional reagents (e.g., templates, second amplification primer,polymerase, nucleotides, cofactors, etc.) may be present in prior toformation of the semi-solid support (e.g., in a liquid prior to gelformation), or one or more of the reagents may be diffused into thesemi-solid support after its formation. The pore size of the semi-solidsupport is selected to allow such diffusion. As is well known in theart, in the case of a polyacrylamide gel, pore size is determined mainlyby the concentration of acrylamide monomer and to a lesser extent by thecrosslinking agent. Similar considerations apply in the case of othersemi-solid support materials. Appropriate cross-linkers andconcentrations to achieve a desired pore size can be selected. Incertain embodiments of the invention an additive such as a cationiclipid, polyamine, polycation, etc., is included in the solution prior topolymerization, which forms in-gel micelles or aggregates surroundingthe microparticles. Methods disclosed in U.S. Pat. Nos. 5,705,628,5,898,071, and 6,534,262 may also be used. For example, various“crowding reagents” can be used to crowd DNA near beads for clonal PCR.SPRI® magnetic bead technology and/or conditions can also be employed.See, e.g., U.S. Pat. No. 5,665,572, demonstrating effective PCRamplification in the presence of 10% polyethylene glycol (PEG). Incertain embodiments of the inventive methods amplification (e.g., PCR),ligation, or both, are performed in the presence of a reagent such asbetaine, polyethylene glycol, PVP-40, or the like. These reagents may beadded to a solution, present in an emulsion, and/or diffused into asemi-solid support.

The semi-solid support may be located or assembled on a substantiallyplanar rigid substrate. In certain preferred embodiments the substrateis transparent to radiation of the excitation and emission wavelengthsused for excitation and detection of typical labels (e.g., fluorescentlabels, quantum dots, plasmon resonant particles, nanoclusters), e.g.,between approximately 400-900 nm. Materials such as glass, plastic,quartz, etc., are suitable. The semi-solid support may adhere to thesubstrate and may optionally be affixed to the substrate using any of avariety of methods. The substrate may or may not be coated with asubstance that enhances adherence or bonding, e.g., silane, polylysine,etc. U.S. Pat. No. 6,511,803 describes methods for synthesizing clonalpopulations of templates using PCR in semi-solid supports, methods forpreparing semi-solid supports on substantially planar substrates, etc.Similar methods may be used in the present invention. The substrate mayhave a well or depression to contain the liquid prior to formation ofthe semi-solid substrate. Alternately, a raised barrier or mask may beused for this purpose.

The above approach provides an alternative to the use of reactors inemulsions to generate spatially localized populations of clonaltemplates. The clonal populations are present at discrete locations inthe semi-solid support, such that a signal can be acquired from eachpopulation during sequencing for purposes of detecting a newly ligatedextension probe, e.g., by imaging. In some embodiments of the invention,two or more distinct clonal populations are amplified from a singlenucleic acid fragment and are present as a mixture at a discretelocation in the semi-solid support. Each of the clonal populations inthe mixture may comprise a tag, e.g., so that the discrete locationcontains fragments containing a 5′ tag and fragments containing a 3′tag. The clonal templates comprising the 5′ tag and the 3′ tag containdifferent sequencing primers, so that they can be sequencedindependently of one another. This approach is identical to the approachdescribed above for producing multiple populations of substantiallyidentical nucleic acids on a microparticle and obtaining sequencinginformation for both members of a paired tag from a singlemicroparticle.

In general, a semi-solid support for use in any of the inventive methodsforms a layer of about 100 microns or less in thickness, e.g., about 50microns thick or less, e.g., between about 20 and 40 microns thick,inclusive. A cover slip or other similar object having a substantiallyplanar surface can be placed atop the semi-solid support material,preferably prior to polymerization, to help produce a uniform gel layer,e.g. to form a gel layer that is substantially planar and/orsubstantially uniform in thickness.

In yet other embodiments of the invention, modifications to the abovemethods are used, in which templates are synthesized by PCR onmicroparticles having a suitable amplification primer attached thereto,wherein the microparticles are immobilized in or on a semi-solid supportprior to template synthesis, i.e., they are fully or partially embeddedin the semi-solid support. Generally the microparticles are completelysurrounded by the semi-solid support material, though they may rest onan underlying substrate. The microparticles thus remain at substantiallyfixed positions with respect to one another unless the semi-solidsupport is disrupted. This approach provides another alternative to theuse of emulsions to generate spatially localized populations of clonaltemplates. Microparticles may be mixed with liquid prior to formation ofthe semi-solid support. Alternatively, microparticles may be arrayed ona substantially planar substrate, and liquid added to the microparticlearray prior to polymerization, crosslinking, etc. The microparticleshave a first amplification primer attached thereto. The secondamplification primer may, but need not be, be attached to the semi-solidsupport. Additional reagents (e.g., template, second amplificationprimer, polymerase, nucleotides, cofactors, etc.) may be present priorto formation of the semi-solid support (e.g., in a liquid prior to gelformation), or one or more of these reagents may be diffused into thesemi-solid support after gel formation. The semi-solid substrate isgenerally formed as described above, e.g., on a glass slide.

In certain embodiments of the invention the gel can be solubilized(e.g., digested or depolymerized or dissolved) so that microparticleswith attached clonal template populations can be conveniently recovered(e.g., by use of a magnet in the case of magnetic particles) followingtemplate synthesis. Gels that can be solubilized, digested,depolymerized, dissolved, etc., are referred to herein as “reversible”.Conventional polyacrylamide polymerization involves the use ofN—N′methylenebisacrylamide (BIS) as a crosslinking agent together with asuitable catalyst to initiate polymerization (e.g.,N,N,N′,N′-tetramethylethylenediamine (TEMED)). To produce a reversiblegel an alternative cross-linking agent such as N—N′ diallyltartardiamide(DATD) may be used. This compound is structurally similar to BIS butpossesses cis-diol groups that can be cleaved by periodic acid, e.g., ina solution containing sodium periodate (Anker, H. S.: F.E.B.S. Lett., 7:293, 1970). Thus DATD gels can be readily solubilized. Gels made usingDATD as the crosslinker are highly transparent and bind well to glassAnother crosslinking agent with DATD-like properties of formingreversible gels is ethylene diacrylate (Choules, G. L. and Zimm, B. S.:Anal. Biochem., 13: 336-339, 1965). N,N′-bisacrylylcystamine (BAC) isanother crosslinker that can be used to form a reversible polyacrylamidegel. Another crosslinking agent that can be used to form gels thatdissolve in periodate is N,N′-(1,2-Dihydroxyethylene)bis-acrylamide(DHEBA). Any of a variety of other materials that form reversiblesemi-solid supports can also be used. For example, thermo-reversiblepolymers such as Pluronics (available from BASF) can be used. Pluronicsare a family of poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymers (Nace, V. M., et al., NonionicSurfactants, Marcel-Dekker, NY, 1996). These materials become semi-solid(gel) at elevated temperatures (e.g., temperatures greater than roomtemperature) and liquify upon cooling. Various methods can be used tochemically derivatize Pluronics, e.g., to facilitate attachment ofprimers thereto (see, e.g., Neff, J. A. et al., J. Biomed. Mater. Res.,40:511, 1998; Prud'homme, R K, et al., Langmuir, 12:4651, 1996).

After solubilization, the microparticles can be collected and subjectedto sequencing using repeated cycles of extension, ligation, andcleavage. Prior to sequencing, the microparticles may be arrayed in oron a second semi-solid support, e.g., at a higher density than that atwhich they were present in or on the first semi-solid support. Thesemi-solid support is typically itself supported by a substantiallyplanar and rigid substrate, e.g., a glass slide.

Thus two general approaches may be used to produce semi-solid supportshaving an array of microparticles bearing clonal template populationsembedded in or on the semi-solid support. The first approach involvesperforming amplification on microparticles that are not present in thesemi-solid support (e.g., by emulsion-based PCR) and then immobilizingthe microparticles in or on a semi-solid support. The second generalapproach involves immobilizing microparticles in or on a semi-solidsupport and then performing amplification. In either case, it may bedesirable to employ procedures to reduce clumping of the microparticlesand/or to align the microparticles substantially in a single focalplane. For example, when immobilizing particles in a polyacrylamide gel,the concentrations of monomer and crosslinker are selected so that theparticles will sink to the bottom of the solution prior to completepolymerization, so that they settle on an underlying planar substrateand are thus arranged in a single plane. In certain embodiments of theinvention an object having a substantially planar surface, such as acover slip, is placed on top of the liquid acrylamide (or other materialcapable of forming a semi-solid support) containing microparticles sothat the acrylamide is trapped between two layers of a “sandwich”structure. The sandwich is then turned over, so that by the action ofgravity the microparticles sink down and rest on the cover slip (orother object having a substantially planar surface). Afterpolymerization, the cover slip is removed. The microparticles are thusembedded in substantially a single plane, close to the surface of thesemi-solid support. (e.g., tangent to the surface).

Rather than immobilizing supports such as microparticles in a semi-solidmatrix as described above, in certain embodiments of the inventionmicroparticles are either covalently or noncovalently attached to asubstantially planar, rigid substrate without use of a semi-solidsupport to immobilize them. A variety of methods for attachingmicroparticles to substrates such as glass, plastic, quartz, silicon,etc., are known in the art. The substrate may or may not be coated(e.g., spin-coated) or functionalized with a material (e.g., any of avariety of polymers) or agent that facilitates attachment. The coatingmay be a thin film, self-assembled monolayer, etc. Either themicroparticles, a moiety attached to the microparticles, oroligonucleotides attached to the microparticles (e.g., the templates)can be attached.

In general, any pair of molecules that exhibit affinity for one anothersuch that they form a binding pair may be used to attach microparticlesor templates to a substrate. The first member of the binding pair isattached covalently or noncovalently to the substrate, and the secondmember of the binding pair is attached covalently or noncovalently tothe microparticles or templates. The first binding partner may beattached to the substrate via a linker. The second binding partner maybe attached to the microparticles or templates via a linker. Forexample, according to one approach, a slide or other suitable substrateis modified with an amine-reactive group (e.g., using a PEG linkercontaining an amine-reactive group). The amine-reactive group reactsunder aqueous conditions (e.g. at pH 8.0) with an amine, e.g., a lysinein any protein, for example, streptavidin. Microparticles functionalizedwith a moiety bearing an amine will therefore become immobilized on thesubstrate. The moiety bearing an amine can be a protein or a suitablyfunctionalized nucleic acid, e.g., a DNA template. Multiple moieties canbe attached to a bead. For example, a bead may have proteins attachedthereto that react with the NHS ester to attach the bead to thesubstrate and may also have DNA templates attached thereto, which can besequenced after the bead is attached to the substrate. Suitably coatedslides bearing a polymer tether having an amine-reactive NHS moiety onone end are commercially available, e.g., from Schott Nexterion, SchottNorth America, Inc., Elmsford, N.Y. 10523). Alternately, coated slides(e.g., biotin-coated slides) are available from Accelr8 TechnologyCorporation, Denver, Colo. Their OptiChem™ technology represents but onemethod for attaching microparticles to a substrate. See, e.g., U.S. Pat.No. 6,844,028. Alternately, microparticles may be attached to asubstrate by functionalising polynucleotides on the bead with biotin by,e.g., the use of terminal transferase with biotin-dideoxyATP and/orbiotin-deoxyATP, and then contacting them with a streptavidin-coatedslide (available from, e.g., Accelr8 Technology Corporation, Denver,Colo.) under conditions which promote a biotin-streptavidin bond.

In general, any of a wide variety of methods known in the art can beused to modify nucleic acids such as oligonucleotide primers, probes,templates, etc., to facilitate the attachment of such nucleic acids tomicroparticles or to other supports or substrates. In addition, any of awide variety of methods known in the art can be used to modifymicroparticles or others supports to facilitate the attachment ofnucleic acids thereto, to facilitate the attachment of microparticles tosupports or substrates, etc. Microspheres are available that havesurface chemistries that facilitate the attachment of a desiredfunctionality. Some examples of these surface chemistries include, butare not limited to, amino groups including aliphatic and aromaticamines, carboxylic acids, aldehydes, amides, chloromethyl groups,hydrazide, hydroxyl groups, sulfonates and sulfates. These groups mayreact with groups present in nucleic acids, or nucleic acids may bemodified by attachment of a reactive group. In addition, a large numberof stable bifunctional groups are well known in the art, includinghomobifunctional and heterobifunctional linkers. See, e.g., PierceChemical Technical Library, available at the Web site having URLwww.piercenet.com (originally published in the 1994-95 Pierce Catalog)and G. T. Hermanson, Bioconjugate Techniques, Academic Press, Inc.,1996. See also U.S. Pat. No. 6,632,655.

Arrays of microparticles formed according to the methods describedherein are generally random. As used herein, the terms“randomly-patterned” or “random” refer to a non-ordered, non-Cartesiandistribution (in other words, not arranged at predetermined points orlocations along the x- and y axes of a grid or at defined ‘clockpositions’, degrees or radii from the center of a radial pattern) ofentities (features) over a support, that is not achieved through anintentional design (or program by which such a design may be achieved)or by placement of individual entities. Such a “randomly-patterned” or“random” array of entities may be achieved by dropping, spraying,plating, spreading, distributing, etc., a solution, emulsion, aerosol,vapor or dry preparation comprising a pool of entities onto or into asupport and allowing them to settle onto or into the support withoutintervention in any manner to direct them to specific sites in or on thesupport. For example, entities may be suspended in a solution thatcontains precursors to a semi-solid support (e.g., acrylamide monomers).The solution is then distributed on a second support and the semi-solidsupport forms on the second support. Entities are embedded in or on thesemi-solid support. Of course non-random arrays can also be used.Generally the methods for forming arrays used herein are distinct frommethods in which, for example, synthesis of a polynucleotide occurs bysequential application of individual nucleotide subunits at predefinedlocations on a substrate.

FIG. 14B (top) shows a fluorescence image of a slide (1 inch by 3 inch)having a polyacrylamide gel thereon. Beads (1 micron diameter) with afluorescently labeled oligonucleotide hybridized to templates attachedto the beads are immobilized in the gel. The image shows a bead surfacedensity (i.e., number of beads per unit area of the substrate, withinthe region where the beads are located) sufficient to imageapproximately 280 million beads per slide. The surface density andimageable area are sufficient to image at least 500 million beads on asingle slide. For example, FIG. 14B (bottom) shows a schematic diagramof a slide with a Teflon® mask surrounding a clear area in which beadsare to be embedded in a semi-solid support layer such as apolyacrylamide gel. The area of this mask is 864 mm². With 500 millionbeads, the surface density is 578,000 beads per mm². A close-packedhexagonal array of 1 micron beads gives 1,155,000 beads per mm², so thisembodiment results in an array having 52% of the theoretical maximumdensity. It will be appreciated that smaller and larger numbers ofbeads, and greater or lesser bead surface densities, can be used than inthis particular embodiment.

Microparticles may be arrayed in or on a substantially planar semi-solidsupport, or on another support or substrate, at a variety of densities,which can be defined in a number of ways. For example, the density maybe expressed in terms of the number of microparticles (e.g., sphericalmicroparticles) per unit area of a substantially planar array. Incertain embodiments of the invention the number of microparticles perunit area of a substantially planar array is at least 80% of the numberof microparticles in a hexagonal array (by “hexagonal array” is meant asubstantially planar array of microparticles in which everymicroparticle in the array contacts at least six other adjacentmicroparticles of equal area as described in U.S. Pat. No. 6,406,848).However, in other embodiments of the invention the microparticle densityis lower, e.g., the number of microparticles per unit area of asubstantially planar array is less than 80%, less than 70%, less than60%, or less than 50% of the number of microparticles in a hexagonalarray. Without wishing to be bound by any theory, it may be preferableto utilize lower densities such as these in order, for example, to allowadequate diffusion of reagents such as enzymes, primers, cofactors,etc., and to avoid a reagent partitioning effect that may occur ifcertain reagents have differential affinity for microparticles or becomeentrapped therein. Such an effect may result in different reactionconditions at different positions on the array and may even preventaccess to certain locations on the array by these reagents. Theseproblems may be exacerbated when reactions are performed in a flow cellsince the reagents move through the flow cell in a directional manner.In certain embodiments of the invention a mixing device, e.g., devicesthat achieve fluid mixing by mechanical or acoustical means, is includedwithin the chamber of a flow cell. A number of suitable mixing devicesare known in the art.

The inventive sequencing methods can be practiced using templatesarranged in array formats of all types, including both random andnonrandom arrays, which can be arrays of microparticles or arrays oftemplates themselves. For example, supports with templates arrayedthereon are described in U.S. Pat. No. 5,641,658 and PCT Pub. No.WO0018957. Arrays may be located on a wide variety of substrates such asfilters, membranes (e.g., nylon), metal surfaces, etc. Additionalexamples of array formats on which sequencing by repeated cycles ofextension, ligation, and cleavage can be performed are arrays of beadslocated in wells at the terminal or distal end of individual opticalfibers in a fiber optic bundle. See, e.g., bead arrays and “arrays ofarrays” described in US publications and patents, e.g., U.S. Pat. Nos.6,023,540; 6,429,027, 20040185483, 2002187515, PCT applicationsUS98/05025, and PCT US98/09163, and PCT publication WO0039587. Beadswith templates attached thereto can be arrayed as described therein.Amplification is preferably performed prior to formation of the array.Arrays formed on such substrates need not necessarily be substantiallyplanar.

In other embodiments, PCR is performed on arrays that compriseoligonucleotides attached to a substrate or support, (see, e.g., U.S.Pat. Nos. 5,744,305; 5,800,992; 6,646,243 and related patents(Affymetrix); PCT publications WO2004029586; WO03065038; WO03040410(Nimblegen)). In general, such oligonucleotides have a free 3′ or 5′end. If desired, the end can be modified, e.g., by adding a phosphategroup or an OH group to a 3′ end if one is not already present. Templatemolecules comprising a region complementary to the oligonucleotideattached to the support or substrate are hybridized to theoligonucleotide, and PCR is performed in situ on the array, resulting ina clonal template population at each location on the array. Theoligonucleotide attached to the array may serve as one of theamplification primers. The templates are then sequenced using theligation-based methods described herein. Sequencing can also beperformed on templates in arrays such as those described in U.S. Pub.No. 20030068629.

Yet other methods for preparation of DNA arrays on surfaces can be used.For example, alkanethiols modified with terminal aldehyde groups canused to prepare a self-assembled monolayer (SAM) on a gold surface. Thealdehyde groups of the monolayer may be reacted with amine-modifiedoligonucleotides or other amine-bearing biomolecules to form a Schiffbase, which may then be reduced to a stable secondary amine by treatmentwith sodium cyanoborohydride (Peelen & Smith, Langmuir, 21(1):266-71,2005). PCR amplification of templates can then be performed.Alternately, microparticles having clonal populations of templatesattached thereto may be attached to surfaces by reacting an amine groupon the microparticle or on templates or oligonucleotides attached to theparticle, with such surfaces.

Still another method of obtaining microparticles with clonal templatepopulations attached thereto is the “solid phase cloning” approachdescribed in U.S. Pat. No. 5,604,097, which makes use of oligonucleotidetags for sorting polynucleotides onto microparticles such that onlypolynucleotides of the same sequence will be attached to any particularmicroparticle.

In certain embodiments of the invention sequencing by repeated cycles ofextension, ligation, and cleavage is performed by diffusing sequencingreagents (e.g., extension probes, ligase, phosphatase, etc.) into asemi-solid support such as a gel having clonal populations of templatesimmobilized in or on the support such that each clonal population islocalized to a spatially distinct region of the support. In certainembodiments the templates are attached directly to the semi-solidsupport as described above. However, in preferred embodiments thetemplates are immobilized on a second support such as a microparticlethat is in turn immobilized in or on the semi-solid support, as alsodescribed above.

As described in Example 1, the inventors have shown that robust ligationand cleavage can be performed on templates attached to beads that areimmobilized in polyacrylamide gels. The invention thus provides a methodof ligating a first polynucleotide to a second polynucleotide comprisingsteps of: (a) providing a first polynucleotide immobilized in or on asemi-solid support; (b) contacting the first polynucleotide with asecond polynucleotide and a ligase; and (c) maintaining the first andsecond polynucleotides in the presence of ligase under suitableconditions for ligation. Suitable conditions include the provision ofappropriate buffers, cofactors, temperature, times, etc., for theparticular ligase being used. In a preferred embodiment the semi-solidsupport is a gel such as an acrylamide gel. In a further preferredembodiment the first polynucleotide is immobilized in or on thesemi-solid support as a result of attachment to a support such as abead, which is itself immobilized in or on the semi-solid support, e.g.,by being partly or completely embedded in the support matrix.Alternately, the first polynucleotide may be attached directly to thesemi-solid support via a linkage such as an acrydite moiety. The linkagemay be covalent or noncovalent (e.g., via a biotin-avidin interaction).U.S. Pat. No. 6,511,803 describes a variety of methods that may be usedto a attach a nucleic acid molecule to a preferred support of theinvention, i.e., a polyacrylamide gel.

The invention further provides a method of cleaving a polynucleotidecomprising steps of: (a) providing a polynucleotide immobilized in or ona semi-solid support, wherein the polynucleotide comprises a scissilelinkage; (b) contacting the polynucleotide with a cleavage agent; and(c) maintaining the polynucleotide in the presence of the cleavage agentunder conditions suitable for cleavage. Suitable conditions include theprovision of appropriate buffers, temperatures, times, etc., for theparticular cleavage agent. In a preferred embodiment the semi-solidsupport is a gel such as an acrylamide gel. In a further preferredembodiment the polynucleotide is immobilized in the semi-solid supportas a result of attachment to a support such as a bead, which is itselfimmobilized in the semi-solid support. Alternately, the polynucleotidemay be attached directly to the semi-solid support via a linkage such asan acrydite moiety. The linkage may be covalent or noncovalent (e.g.,via a biotin-avidin interaction).

Macevicz discloses sequencing a single template species having aparticular sequence. He does not discuss the possibility of performinghis method in parallel to simultaneously sequence a plurality oftemplates having different sequences. The inventors have recognized thatin order to efficiently perform sequencing in a high throughput manner,it is desirable to prepare a plurality of supports (e.g., beads), asdescribed above, such that each support has templates of a particularsequence attached thereto, and to perform the methods described hereinsimultaneously on templates attached to each support. In certainembodiments of this approach, a plurality of such supports are arrayedin or on a planar substrate such as a slide. In certain embodiments thesupports are arrayed in or on a gel. The supports may be arrayed in arandom fashion, i.e., the location of each support on the substrate isnot predetermined. The supports need not be located at regularly spacedintervals or positioned in an ordered arrangement of rows and columns,etc. Preferably the supports are arrayed at a density such that it ispossible to detect an individual signal from many or most of thesupports. In certain preferred embodiments the supports are primarilydistributed in a single focal plane. Multiple supports having templatesof the same sequence attached thereto may be included, e.g., forpurposes of quality control. Sequencing reactions are performed inparallel on templates attached to each of the supports.

Signals may be collected using any of a variety of means, includingvarious imaging modalities. Preferably, for embodiments in whichsequencing is performed on microparticles that are arrayed on asubstrate (e.g., beads embedded in a semi-solid support positioned on asubstrate) prior to detection, the imaging device has a resolution of 1μm or less. For example, a scanning microscope fitted with a CCD camera,or a microarray scanner with sufficient resolution could be used.Alternately, beads can be passed through a flow cell or fluidicsworkstation attached to a microscope equipped for fluorescencedetection. Other methods for collecting signal include fiber opticbundles. Appropriate image acquisition and processing software may beused.

In certain embodiments of the invention sequencing is performed in amicrofluidic device. For example, beads with attached templates may beloaded into the device and reagents flowed therethrough. Templatesynthesis, e.g., using PCR, can also be performed in the device. U.S.Pat. No. 6,632,655 describes an example of a suitable microfluidicdevice.

D. Sequencing with Re-initialization Using Different InitializingOligonucleotides

In a preferred embodiment of the instant invention, the extended strandgenerated by extending a first initializing oligonucletide is removedfrom the template following a sufficient number of cycles and a secondinitializing oligonucleotide is annealed to the binding region, followedby cycles of extension, ligation, and detection. The process is repeatedwith any number of different initializing oligonucleotides. Inembodiments in which the extension probes are cleaved, preferably thenumber of different initializing oligonucleotides used (and thus thenumber of reactions) equals the length of the portion of the extensionprobe that remains hybridized to the template following release of thedistal portion of the probe. Thus according to this embodiment sequenceinformation (e.g., the order and identity of each nucleotide) can beobtained from the templates that are attached to a single support whilestill reading deep into the sequence using substantially fewer cyclesthan would be required if successive nucleotides were identified in eachcycle.

Embodiments in which the initializing oligonucleotides are boundsequentially to the same template have certain advantages over anapproach that requires dividing the template into multiple aliquots,such as the methods taught by Macevicz. For example, applying theinitializing oligonucleotides to the same template avoids the need tokeep track of, and later, combine data acquired from multiple aliquots.In embodiments in which the supports are arrayed in a random fashionsuch that the position of individual supports is not predetermined, itwould be difficult or impossible to reliably combine partial sequenceinformation from multiple supports each of which had templates of thesame sequence attached thereto.

E. Identification of Multiple Nucleotides in Each Cycle on a SingleTemplate

Macevicz teaches identification of single nucleotides in the template ineach cycle of extension, ligation, and detection. However, the inventorshave recognized that the methods may be modified to allow identificationof multiple nucleotides in the template in each cycle. In this case theextension probes are labeled so that the identity of two or more,preferably contiguous, nucleotides abutting the extended duplex can bedetermined from the label. In other words, the sequence determiningportion of the extension probes is more than a single nucleotide andtypically comprises the proximal nucleotide, the immediately adjacentnucleotide, and possibly one or more additional, preferably contiguousnucleotides, all of which hybridize specifically to the template. Forexample, rather than using 4 labels to identify the bases A, G, C, andT, 16 distinguishably labeled probes or probe combinations are used toidentify the 16 possible dinucleotides AA, AG, AC, AT, GA, GG, GC, GT,CA, CG, CC, CT, TA, TG, TC, and TT. The sequence determining portion ofeach distinguishably labeled extension probe is complementary to one ofthese dinucleotides. Similar methods utilizing more labels allowidentification of longer nucleotide sequences in each cycle.

F. Labels

The term “label” is used herein in a broad sense to denote anydetectable moiety or plurality of detectable moieties attached to orassociated with a probe, by which probes of different species (e.g.,probes with different terminal nucleotides) may be distinguished fromone another. Thus there need not be a one to one correspondence betweena label and a specific detectable moiety. For example, multipledetectable moieties can be attached to a single probe, resulting in acombined signal that allows the probe to be distinguished from probeshaving a different detectable moiety or set of detectable moietiesattached thereto. For example, combinations of detectable moieties canbe used in accordance with a labeling scheme referred to as“Combinatorial Multicolor Coding”, which is described in U.S. Pat. No.6,632,609 and in Speicher, et al., Nature Genetics, 12:368-375, 1996.

The probes of the invention can be labeled in a variety of ways,including the direct or indirect attachment of fluorescent orchemiluminescent moieties, colorimetric moieties, enzymatic moietiesthat generate a detectable signal when contacted with a substrate, andthe like. Macevicz teaches that the probes may be labeled withfluorescent dyes, e.g. as disclosed by Menchen et al, U.S. Pat. No.5,188,934; Begot et al PCT application PCT/US90105565. The terms“fluorescent dye”, and “fluorophore” as used herein refer to moietiesthat absorb light energy at a defined excitation wavelength and emitlight energy at a different wavelength. Preferably the labels selectedfor use with a given mixture of probes are spectrally resolvable. Asused herein, “spectrally resolvable” means that the labels may bedistinguished on the basis of their spectral characteristics,particularly fluorescence emission wavelength, under conditions ofoperation. For example, the identity of the one or more terminalnucleotides may be correlated to a distinct wavelength of maximum lightemission intensity, or perhaps a ratio of intensities at differentwavelengths. The spectral characteristic(s) of a label that is/are usedto detect and identify a label is referred to as a “color” herein. Itwill be appreciated that a label is frequently identified on the basisof a specific spectral characteristic, e.g., the frequency of maximumemission intensity in the case of labels that consist of a singledetectable moiety, or the frequencies of emission peaks in the case oflabels that consist of multiple detectable moieties.

Preferably, four probes are provided that allow a one-to-onecorrespondence between each of four spectrally resolvable fluorescentdyes and the four possible terminal nucleotides of the probes. Sets ofspectrally resolvable dyes are disclosed in U.S. Pat. Nos. 4,855,225 and5,188,934; International application PCT/US90/05565; and Lee et al,Nucleic Acids Researchs, 20: 2471-2483 (1992). In certain embodiments aset consisting of FITC, HEX™, Texas Red, and Cy5 is preferred. Numeroussuitable fluorescent dyes are commercially available, e.g., fromMolecular Probes, Inc., Eugene Oreg. Specific examples of fluorescentdyes include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680),AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665), CAL dyes, Carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cyanine dyes(Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Oyster dyes, Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X. See TheHandbook of Fluorescent Probes and Research Products, 9^(th) ed.,Molecular Probes, Inc., for further description.

Rather than being directly detectable themselves, some fluorescentgroups transfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal. The use of quenchers, i.e., is also withinthe scope of the invention. The term “quencher” refers to a moiety thatis capable of absorbing the energy of an excited fluorescent label whenlocated in close proximity and of dissipating that energy without theemission of visible light. Examples of quenchers include, but are notlimited to DABCYL (4-(4′-dimethylaminophenylazo)benzoic acid)succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester(QSY-7), and 4′,5′-dinitrofluorescein carboxylic acid, succinimidylester (QSY-33) (all available from Molecular Probes), quencher1 (Q1;available from Epoch), or “Black hole quenchers” BHQ-1, BHQ-2, and BHQ-3(available form BioSearch, Inc.).

In addition to the various detectable moieties mentioned above, thepresent invention also comprehends use of spectrally resolvable quantumdots, metal nanoparticles or nanoclusters, etc., which may either bedirectly attached to an oligonucleotide probe or may be embedded in orassociated with a polymeric matrix which is then attached to the probe.As mentioned above, detectable moieties need not themselves be directlydetectable. For example, they may act on a substrate which is detected,or they may require modification to become detectable.

As described above, in certain embodiments of the invention a labelconsists of a plurality of detectable moieties. The combined signal fromthese detectable moieties produces a color that is used to identify theprobe. For example, a “purple” probe of a particular sequence could beconstructed by attaching “blue” and “red” detectable moieties thereto.Alternatively, a distinct color can be generated by combining twospecies of probe having the same sequence but labeled with differentdetectable moieties to produce a mixed probe. Thus a “purple” probe of aparticular sequence can be produced by constructing two species of probehaving that sequence. “Red” detectable moieties are attached to thefirst species, and “blue” detectable moieties are attached to the secondspecies. Aliquots of these two species are mixed. Various shades ofpurple can be produced by mixing aliquots in different ratios. Thisapproach offers a number of advantages. Firstly, it allows theproduction of multiple distinguishable probes using a smaller number ofdetectable moieties. Secondly, using a mixed probe can provide a degreeof redundancy that may help reduce bias that may result frominteractions between particular detectable moieties and particularnucleotides.

In certain embodiments of the invention a detectable moiety is attachedto a nucleotide in an oligonucleotide extension probe by a cleavablelinkage, which allows removal of the detectable moiety followingligation and detection. Any of a variety of different cleavable linkagesmay be used. As used herein, the term “cleavable linkage” refers to achemical moiety that joins a detectable moiety to a nucleotide, and thatcan be cleaved to remove the detectable moiety from the nucleotide whendesired, essentially without altering the nucleotide or the nucleic acidmolecule it is attached to. Cleavage may be accomplished, for example,by acid or base treatment, or by oxidation or reduction of the linkage,or by light treatment (photocleavage), depending upon the nature of thelinkage. Examples of cleavable linkages and cleavage agents aredescribed in Shirnkus et al., 1985, Proc. Natl. Acad. Sci. USA 82:2593-2597; Soukup et al., 1995, Bioconjug. Chem. 6: 135-138; Shimikus etal., 1986, DNA 5: 247-255; and Herman and Fenn, 1990, Meth. Enzymol.184: 584-588.

For example, as described in U.S. Pat. No. 6,511,803, a disulfidelinkage can be reduced and thereby cleaved using thiol compound reducingagents such as dithiothreitol (DTT). Fluorophores are available with asulfhydryl (SH) group available for conjugation (e.g., Cyanine 5 orCyanine 3 fluorophores with SH groups; New England Nuclear—DuPont), asare nucleotides with a reactive aryl amino group (e.g., dCTP). Areactive pyridyldithiol will react with a sulfhydryl group to give asulfhydryl bond that is cleavable with reducing agents such asdithiothreitol. An NHS-ester heterobifunctional crosslinker (Pierce) canbe used to link a deoxynucleotide comprising a reactive aryl amino groupto a pyridyldithiol group, which is in turn reactive with the SH on afluorophore, to yield a disulfide bonded, cleavablenucleotide-fluorophore complex useful in the methods of the invention.Alternatively, a cis-glycol linkage between a nucleotide and afluorophore can be cleaved by periodate. A variety of cleavable linkagesare described in U.S. Pat. Nos. 6,664,079, and 6,632,655, US PublishedApplication 20030104437, WO 04/18497 and WO 03/48387.

In other embodiments of the invention a detectable moiety that can berendered nondetectable by exposure to electromagnetic energy such aslight (photobleaching) is used.

In those embodiments of the invention that employ extension probeshaving a label that is attached to the probe by a cleavable linkage, orhaving a label that can be photobleached, a the sequencing methods willtypically include a step of cleavage or photobleaching in one or morecycles after ligation and label detection have been performed. Asmentioned above, cleavage of the scissile linkage present in theoligonucleotide extension probes may not proceed to completion (i.e.,less than 100% of the newly ligated probes may be cleaved in the cyclein which they were ligated). Since such probes generally comprise anon-extendable terminus, or are capped, they will not contribute tosuccessive cycles. However, failure to cleave the probe means that thelabel remains associated with the template molecule to which the probeligated, which contributes background signal (i.e., backgroundfluorescence) that can increase the noise in subsequent cycles.Incorporating a step of cleavage or photobleaching to remove the labelor render it undetectable reduces this background and improves thesignal to noise ratio. Cleavage or photobleaching can be performed asoften as every cycle, or less frequently, such as every other, everythird, or every fifth or more cycles. In certain embodiments of theinvention it is not necessary to actually add any additional steps toachieve cleavage of the cleavable linker. For example, a cleavage agentsuch as DTT may already be present in a wash buffer that may be used toremove unligated extension probes.

G. Preferred Scissile Linkages

The inventors have discovered that extension probes having at least onephosphorothiolate linkage are particularly useful in the practice ofmethods for sequencing by successive cycles of extension, ligation,detection, and cleavage. In such linkages one of the bridging oxygenatoms of a phosphodiester bond is replaced by a sulfur atom. Thephosphorothiolate linkage can be either a 5′-S-phosphorothiolate linkage(3′-O—P—S-5′) as shown in FIG. 4A or a 3′-S-phosphorothiolate linkage(3′-S—P—O-5′) as shown in FIG. 4B. It is to be understood that thephosphorus atom in linkages represented as 3′-O—P—S-5′ or 3′-S—P—O-5′may be attached to two non-bridging oxygen atoms as shown in FIGS. 4Aand 4B (as in typical phosphodiester bonds). Alternately, the phosphorusatom could be attached to any of a variety of other atoms or groups,e.g., S, CH₃, BH₃, etc. Thus one aspect of the invention is labeledolignucleotide probes comprising phosphorothiolate linkages. While theprobes find particular use in the sequencing methods described herein,they may also be used for a variety of other purposes. In particular,the invention provides (i) an oligonucleotide of the form5′-O—P—O—X—O—P—S—(N)_(k)N_(B)*-3′; and (ii) an oligonucleotide of theform 5′-N_(B)*(N)_(k)—S—P—O—X-3′. In each of these probes N representsany nucleotide, N_(B) represents a moiety that is not extendable byligase, * represents a detectable moiety, X represents a nucleotide, andk is between 1 and 100. In certain embodiments k is between 1 and 50,between 1 and 30, between 1 and 20, e.g., between 4 and 10, with theproviso that a detectable moiety may be present on any nucleotide of(N)_(k) instead of, or in addition to, N_(B). The terminal nucleotidesin any of these probes may or may not include a phosphate group or ahydroxyl group. Furthermore, it will be appreciated that the phosphorusatoms will generally be attached to two additional (non-bridging) oxygenatoms in preferred embodiments.

Methods for synthesizing oligonucleotides containing 5′-Sphosphorothiolate or 3′-S-phosphorothiolate linkages are known in theart, and certain of these methods are amenable to automated solid phaseoligonucleotide synthesis. Synthesis procedures are described, forexample, in Cook, A F, J. Am. Chem. Soc., 92:190-195, 1970; Chladek, S.et al., J. Am. Chem. Soc., 94:2079-2084, 1972; Rybakov, V N, et al.,Nucleic Acids Res., 9:189-201, 1981; Cosstick, R. and Vyle, J S, J.Chem. Soc. CHem. Commun., 992-992, 1988; Mag, M., et al., Nucleic AcidsRes., 19(7); 1437-1441, 1991; Xu, Y, and Kool, E T, Nucleic Acids Res.,26(13): 3159-3164, 1998; Cosstick, R. and Vyle, J S, Tetrahedron Lett.,30:4693-4696, 1989; Cosstick, R. and Vyle, J S, Nucleic Acids Res.,18:829-835, 1990; Sun, S G and Piccirilli, J A, Nucl. Nucl.,16:1543-1545, 1997; Sun S G, et al., RNA, 3:1352-1363, 1997; Vyle, J S,et al., Tetrahedron Lett., 33:3017-3020, 1992; Li, X., et al., J. Chem.Soc. Perkin Trans., 1:2123-22129, 1994; Liu, X H and Reese, C B,Tetrahedron Lett., 37: 925-928, 1996; Weinstein, L B, et al., J. Am.Chem. Soc., 118:10341-10350, 1996; and Sabbagh, G., et al., NucleicAcids Res., 32(2):495-501, 2004. In addition, the present inventors havedeveloped new synthesis methods. For example, FIG. 7 shows a synthesisscheme for a 3′-phosphoroamidite of dA. A similar scheme may be used forsynthesis of a 3′-phosphoroamidite of dG. These phosphoroamidites may beused to synthesize oligonucleotides containing 3′-S-phosphorothiolatelinkages associated with purine nucleosides, e.g., using an automatedDNA synthesizer.

Phosphorothiolate linkages can be cleaved using a variety ofmetal-containing agents. The metal can be, for example, Ag, Hg, Cu, Mn,Zn or Cd. Preferably the agent is a water-soluble salt that providesAg⁺, Hg⁺⁺, Cu⁺⁺, Mn⁺⁺, Zn⁺ or Cd⁺ anions (salts that provide ions ofother oxidation states can also be used). I₂ can also be used.Silver-containing salts such as silver nitrate (AgNO₃), or other saltsthat provide Ag⁺ ions, are particularly preferred. Suitable conditionsinclude, for example, 50 mM AgNO₃ at about 22-37° C. for 10 minutes ormore, e.g., 30 minutes. Preferably the pH is between 4.0 and 10.0, morepreferably between 5.0 and 9.0, e.g., between about 6.0 and 8.0, e.g.,about 7.0. See, e.g., Mag, M., et al., Nucleic Acids Res.,19(7):1437-1441, 1991. An exemplary protocol is provided in Example 1.

Sequencing in the 5′→3′ direction may be performed using extensionprobes containing a 3′-O—P—S-5′ linkage. FIG. 5A shows a single cycle ofhybridization, ligation, and cleavage using an extension probe of theform 5′-O—P—O—X—O—P—S—NNNNN_(B)*-3′ where N represents any nucleotide,N_(B) represents a moiety that is not extendable by ligase (e.g., N_(B)is a nucleotide that lacks a 3′ hydroxyl group or has an attachedblocking moiety), * represents a detectable moiety, and X represents anucleotide whose identity corresponds to the detectable moiety.Alternately, any of a large number of blocking moieties can be attachedto the 3′ terminal nucleotide to prevent multiple ligations. Forexample, attaching a bulky group to the sugar portion of the nucleotide,e.g., at the 2′ or 3′ position, will prevent ligation. A fluorescentlabel may serve as an appropriate bulky group.

A template containing binding region 40 and polynucleotide region 50 ofunknown sequence is attached to a support, e.g., a bead. In a preferredembodiment, as shown in FIG. 5A, the binding region is located at theopposite end of the template from the point of attachment to thesupport. An initializing oligonucleotide 30 with an extendable terminus(in this case a free 3′ OH group) is annealed to binding region 40.Extension probe 60 is hybridized to the template in polynucleotideregion 50. Nucleotide X forms a complementary base pair with unknownnucleotide Y in the template. Extension probe 60 is ligated to theinitializing oligonucleotide (e.g., using T4 ligase). Followingligation, the label attached to extension probe 60 is detected (notshown). The label corresponds to the identity of nucleotide X. Thusnucleotide Y is identified as the nucleotide complementary to nucleotideX. Extension probe 60 is then cleaved at the phosphorothiolate linkage(e.g., using AgNO₃ or another salt that provides Ag⁺ ions), resulting inan extended duplex. Cleavage leaves a phosphate group at the 3′ end ofthe extended duplex. Phosphatase treatment is used to generate anextendable probe terminus on the extended duplex. The process isrepeated for a desired number of cycles.

In a preferred embodiment sequencing is performed in the 3′→5′ directionusing extension probes containing a 3′-S—P—O-5′ linkage. FIG. 5B shows asingle cycle of hybridization, ligation, and cleavage using an extensionprobe of the form 5′-N_(B)*—NNNN—S—P—O—X-3′ where N represents anynucleotide, N_(B) represents a moiety that is not extendable by ligase(e.g., N_(B) is a nucleotide that lacks a 5′ phosphate group or has anattached blocking moiety), * represents a detectable moiety, and Xrepresents a nucleotide whose identity corresponds to the detectablemoiety.

A template containing binding region 40 and polynucleotide region 50 ofunknown sequence is attached to a support, e.g., a bead. In a preferredembodiment, as shown in FIG. 5B, the binding region is located at theopposite end of the template from the point of attachment to thesupport. An initializing oligonucleotide 30 with an extendable terminus(in this case a free 5′ phosphate group) is annealed to binding region40. Extension probe 60 is hybridized to the template in polynucleotideregion 50. Nucleotide X forms a complementary base pair with unknownnucleotide Y in the template. Extension probe 60 is ligated to theinitializing oligonucleotide (e.g., using T4 ligase). Followingligation, the label attached to extension probe 60 is detected (notshown). The label corresponds to the identity of nucleotide X. Thusnucleotide Y is identified as the nucleotide complementary to nucleotideX. Extension probe 60 is then cleaved at the phosphorothiolate linkage(e.g., using AgNO₃ or another salt that provides Ag⁺ ions), resulting inan extended duplex. Cleavage leaves an extendable monophosphate group atthe 5′ terminus of the extended duplex and it is therefore unnecessaryto perform an additional step to generate an extendable terminus. Theprocess is repeated for a desired number of cycles.

It will be appreciated that a number of variations of this scheme can beused. For example, the probe may be shorter or longer than 6nucleotides; the label need not be on the 3′ terminal nucleotide; theP—S linkage can be located between any two adjacent nucleotides, etc. Inthe embodiments described above, successive cycles of extension,ligation, detection, and cleavage, result in identification ofadjacently located nucleotides. However, by placing the P—S linkagecloser to the distal end of the extension probe (i.e., the end oppositeto that at which ligation occurs), the nucleotides that are sequentiallyidentified will be spaced at intervals along the template, as describedabove and shown in FIGS. 1 and 6.

FIG. 6A-6F is a more detailed diagrammatic illustration of severalsequencing reactions performed sequentially on a single template.Sequencing is performed in the 3′→5′ direction using extension probescontaining 3′-S—P—O-5′ linkages. Each sequencing reaction comprisesmultiple cycles of extension, ligation, detection, and cleavage. Thereactions utilize initializing oligonucleotides that bind to differentportions of the template. The extension probes are 8 nucleotides inlength and contain phosphorothiolate linkages located between the 6^(th)and 7^(th) nucleotides counting from the 3′ end of the probe.Nucleotides 2-6 serve as a spacer such that each reaction allows theidentification of a plurality of nucleotides spaced at intervals alongthe template. By performing multiple reactions in series andappropriately combining the partial sequence information obtained fromeach reaction, the complete sequence of a portion of the template isdetermined.

FIG. 6A shows initialization using a first initializing oligonucleotide(referred to as a primer in FIGS. 6A-6F) that is hybridized to anadapter sequence (referred to above as a binding region) in the templateto provide an extendable duplex. FIGS. 6B-6D show several cycles ofnucleotide identification in which every 6^(th) base of the template isread. In FIG. 6B, a first extension probe having a 3′ terminalnucleotide complementary to the first unknown nucleotide in the templatesequence binds to the template and is ligated to the extendable terminusof the primer. The label attached to the extension probe identifies theprobe as having an A as the 3′ terminal nucleotide and thus identifiesthe first unknown nucleotide in the template sequence as A. FIG. 6Cshows cleavage of the extension oligonucleotide at the phosphorothiolatelinkage with AgNO₃ and release of a portion of the extension probe towhich a label is attached. FIG. 6D shows additional cycles of extension,ligation, and cleavage. Since the probes contain a spacer 5 nucleotidesin length, the sequencing reaction identifies every 6^(th) nucleotide inthe template.

Following a desired number of cycles the extended strand, including thefirst initializing oligonucleotide, is removed and a second initializingoligonucleotide that binds to a different portion of the binding regionfrom that at which the first initializing oligonucleotide bound, ishybridized to the template. FIG. 6E shows a second sequencing reactionin which initialization is performed with a second initializingoligonucleotide, followed by several cycles of nucleotideidentification. FIG. 6F shows initialization using a third initializingoligonucleotide followed by several cycles of nucleotide identification.Extension from the second initializing oligonucleotide allowsidentification of every 6^(th) base in a different “frame” from thenucleotides identified in the first sequencing reaction.

Although extension probes comprising phosphorothiolate linkages arepreferred in certain embodiments of the invention, a variety of otherscissile linkages may be advantageously employed. For example, a largenumber of variations on the O—P—O linkage found in naturally occurringnucleic acids are known (see, e.g., Micklefield, J. Curr. Med. Chem.,8:1157-1179, 2001). Any structures described therein that contain a P—Obond can be modified to contain a scissile P—S bond. For example, anNH—P—0 bond can be changed to an NH—P—S bond.

In some embodiments of the invention the extension probes comprise atrigger residue that renders the nucleic acid susceptible to cleavage bya cleavage agent or combination thereof, optionally followingmodification of the trigger residue by a modifying agent. In particular,the inventors have discovered that enzymes involved in DNA repair areadvantageous cleavage reagents for use in the practice of methods forsequencing by successive cycles of extension, ligation, detection, andcleavage. In general, the presence of a trigger residue such as adamaged base or abasic residue in an extension probe may render theprobe susceptible to cleavage by one or more DNA repair enzymes,optionally following modification by a DNA glycosylase. Thus extensionprobes comprising linkages that are substrates for cleavage by enzymesinvolved in DNA repair such as AP endonucleases are of use in theinvention. Extension probes containing residues that are substrates formodification by enzymes involved in DNA repair, such as DNAglycosylases, wherein the modification renders the probe susceptible tocleavage by an AP endonuclease, are also of particular use in theinvention. In some embodiments the extension probe comprises an abasicresidue, i.e., it lacks a purine or pyrimidine base. The linkage betweenthe abasic residue and an adjacent nucleoside is susceptible to cleavageby an AP endonuclease and is therefore a scissile linkage. In certainembodiments of the invention the abasic residue comprises 2′deoxyribose. In some embodiments the extension probe comprises a damagedbase. The damaged base is a substrate for an enzyme that removes damagedbases, such as a DNA glycosylase. Following removal of the damaged base,the linkage between the resulting abasic residue and an adjacentnucleoside is susceptible to cleavage by an AP endonuclease and istherefore considered a scissile linkage in accordance with theinvention.

Many different AP endonucleases are of use as cleavage reagents in thepresent invention. Two major classes of AP endonuclease have beendistinguished on the basis of the mechanism by which they cleavelinkages adjacent to abasic residues. Class I AP endonucleases, such asendonuclease III (Endo III) and endonuclease VIII (Endo VIII) of E. coliand the human homologs hNTH1, NEIL1, NEIL2, and NEIL3, are AP lyasesthat cleave DNA on the 3′ side of the AP residue, resulting in a 5′portion that has a 3′ terminal phosphate and a 3′ portion that bears a5′ terminal phosphate. Class II AP endonucleases such as endonuclease IV(Endo IV) and exonuclease III (Exo III) of E. coli cleave the DNA 5′ ofthe AP site, which produces a 3′ OH and 5′ deoxyribose phosphate moietyat the termini of the resulting fragments. See, e.g., Doublie, S., etal., Proc. Natl. Acad. Sci. 101(28), 10284-10289, 2004; Haltiwanger, B.M., et al, Biochem J., 345, 85-89, 2000; Levin, J. and Demple, B., Nucl.Acids. Res, 18(17), 1990, and references in all of the foregoing forfurther discussion of various Class I and Class II AP endonucleases andconditions under which they remove damaged bases from DNA and/or cleaveDNA containing an abasic residue. One of ordinary skill in the art willappreciate that a variety of homologs of these enzymes exist in otherorganisms (e.g., yeast) and are of use in the present invention.

Certain enzymes are bifunctional in that they possess both glycosylaseactivity that removes a damaged base to generate an AP residue and alsodisplay a lyase activity that cleaves the phosphodiester backbone 3′ tothe AP site generated by the glycosylase activity. Thus these dualactivity enzymes are both AP endonucleases and DNA glycosylases. Forexample, Endo VIII acts as both an N-glycosylase and an AP-lyase. TheN-glycosylase activity releases damaged pyrimidines from double-strandedDNA, generating an apurinic (AP site). The AP-lyase activity cleaves 3′and 5′ to the AP site leaving a 5′ phosphate and a 3′ phosphate. Damagedbases recognized and removed by Endonuclease VIII include urea,5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydanton, uracilglycol, 6-hydroxy-5,6-dihydrothymine and methyltartronylurea. See, e.g.,Dizdaroglu, M., et al., Biochemistry, 32, 12105-12111, 1993 and Hatahet,Z. et al., J. Biol. Chem., 269, 18814-18820, 1994; Jiang, D., et al., J.Biol. Chem., 272(51), 32220-32229, 1997; Jiang, D., et al., J. Bact.,179(11), 3773-3782, 1997.

Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as8-oxoguanine DNA glycosylase) also acts both as a N-glycosylase and anAP-lyase. The N-glycosylase activity releases damaged purines fromdouble stranded DNA, generating an apurinic (AP site). The AP-lyaseactivity cleaves both 3′ and 5′ to the AP site thereby removing the APsite and leaving a 1 base gap. Some of the damaged bases recognized andremoved by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoguanine),8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxinB1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil. See, e.g.,Tchou, J. et al. J. Biol. Chem., 269, 15318-15324, 1994; Hatahet, Z. etal. J. Biol. Chem., 269, 18814-18820, 1994; Boiteux, S., et al, EMBO J.,5, 3177-3183, 1987; Jiang, D., et al., J. Biol. Chem., 272(51),32220-32229, 1997; Jiang, D., et al., J. Bact., 179(11), 3773-3782,1997.

A number of DNA glycosylases and AP endonucleases are commerciallyavailable, e.g., from New England Biolabs, Ipswich, Mass.

In some embodiments of the invention extension probes comprising a sitethat is a substrate for cleavage by an AP endonuclease are used in thesequencing method as described above for extension probes containing aphosphorothiolate linkage or in sequencing methods AB (see below). Inany of these methods, following ligation of an extension probe to agrowing nucleic acid strand, the extension probe is cleaved using an APendonuclease to remove the portion of the probe that comprises a label.

Depending on the particular AP endonuclease, and depending on whethersequencing is performed in the 3′→5′ or the 5′→3′ direction, it may benecessary or desirable to treat the extended duplex with apolynucleotide kinase or a phosphatase following cleavage in order togenerate an extendable probe terminus on the extended duplex (see FIGS.5A and 5B for depiction of extendable probe termini). Thus in certainmethods of the invention an extendable terminus is generated bytreatment with a polynucleotide kinase or phosphatase. One of ordinaryskill in the art will appreciate that appropriate buffers will beemployed for the various enzymes, and additional steps of washing may beincluded to remove enzymes and provide appropriate conditions forsubsequent steps in the methods.

In other embodiments the extension probe comprises a damaged base thatis a substrate for removal by a DNA glycosylase. A wide range ofcytotoxic and mutagenic DNA bases are removed by different DNAglycosylases, which initiate the base excision repair pathway followingdamage to DNA (Krokan, H. E., et al., Biochem J., 325 (Pt 1):1-16,1997). DNA glycosylases cleave the N-glycosydic bond between the damagedbase and deoxyribose, thus releasing a free base and leaving anapurinic/apyrimidinic (AP) site. In some embodiments the extension probecomprises a uracil residue, which is removed by a uracil-DNA glycosylase(UDG). UDGs are found in all living organisms studied to date, and alarge number of these enzymes are known in the art and are of use inthis invention (Frederica, et al, Biochemistry, 29, 2353-2537, 1990;Krokan, supra). For example, mammalian cells contain at least 4 types ofUDG: mitochondrial UNG1 and nuclear UNG2, SMUG1, TDG, and MBD4 (Krokan,et al., Oncogene, 21, 8935-8948, 2002). UNG1 and UNG2 belong to a highlyconserved family typified by E. coli Ung. In embodiments in which theextension probe comprises a damaged base, following ligation of theextension probe to an extendable probe terminus, the extended duplex iscontacted with a glycosylase that removes the damaged base, therebyproducing an abasic residue. An extension probe that comprises a damagedbase that is subject to removal by a glycosylase is considered to be“readily modifiable to comprise a scissile linkage”. The extended duplexis then contacted with an AP endonuclease, which cleaves a linkagebetween the abasic residue and an adjacent nucleoside, as describedabove. In certain embodiments of the invention a dual activity enzymethat is both a DNA glycosylase and an AP endonuclease is used to performboth of these reactions. In some embodiments the extended duplexcontaining a damaged base is contacted with a DNA glycosylase and an APendonuclease. The enzymes can be used in combination or sequentially(i.e., glycosylase followed by endonuclease) in various embodiments ofthe invention.

In some embodiments of the invention an extension probe comprises atrigger residue which is deoxyinosine. As noted above, E. coliEndonuclease V (Endo V), also called deoxyinosine 3′ endonuclease, andhomologs thereof cleave a nucleic acid containing deoxyinosine at thesecond phosphodiester bond 3′ to the deoxyinosine residue, leaving a 3′OH and 5′ phosphate termini. Thus this bond serves as a scissile linkagein the extension probe. Endo V and its cleavage properties are known inthe art (Yao, M. and Kow Y. W., J. Biol. Chem., 271, 30672-30673 (1996);Yao, M. and Kow Y. W., J. Biol. Chem., 270, 28609-28616 (1995); He, B,et al., Mutat. Res., 459, 109-114 (2000). In addition to deoxyinosine,Endo V also recognizes deoxyuridine, deoxyxanthosine, and deoxyoxanosine(Hitchcock, T. et al., Nuc. Acids Res., 32(13), 32(13) (2004). Mammalianhomologs such as mEndo V also exhibit cleavage activity (Moe, A., etal., Nuc. Acids Res., 31(14), 3893-3900 (2004). While Endo V is apreferred cleavage agent for probes comprising deoxyinosine, othercleavage reagents may also be used to cleave probes comprisingdeoxyinosine. For example, as a damaged base, hypoxanthine may besubject to removal by an appropriate DNA glycosylase, and the resultingextension probe containing an abasic residue is then subject to cleavageby an endonuclease.

It will be appreciated that if deoxyinosine is used as a triggerresidue, it may be desirable to avoid using deoxyinosine elsewhere inthe probe, particularly at positions between the terminus that will beligated to the extendable probe terminus and the trigger residue. Thusif the probe comprises one or more universal bases, a nucleoside otherthan deoxyinosine may be used. It will also be appreciated that where atrigger residue that renders a nucleic acid containing the triggerresidue susceptible to cleavage by a particular cleavage agent is usedin an extension probe, it may be desirable to avoid including otherresidues in the probe (or in other probes that would be used in asequencing reaction together with that extension probe) that wouldtrigger cleavage by the same cleavage agent.

The present invention encompasses the use of any enzyme that cleaves anucleic acid that comprises a trigger residue. Additional enzymes may beidentified by perusing the catalog of enzyme suppliers such as NewEngland Biolabs®, Inc. The New England Biolabs Catalog, 2005 edition(New England Biolabs, Ipswich, Mass. 01938-2723) is incorporated hereinby reference, and the present invention contemplates use of any enzymedisclosed therein that cleaves a nucleic acid containing a triggerresidue, or a homolog of such an enzyme. Other enzymes of use include,e.g., hOGG1 and homologs thereof (Radicella, J P, et al., Proc Natl AcadSci USA., 94(15):8010-5, 1997).

Methods for synthesizing oligonucleotides containing a trigger residuesuch as a damaged base, abasic residue, etc. are known in the art.Methods for synthesizing oligonucleotides containing site that is asubstrate for an AP endonuclease, e.g., oligonucleotides containing anabasic residue are known in the art and are generally amenable toautomated solid phase oligonucleotide synthesis. In some embodiments anoligonucleotide containing uridine at the desired location of the abasicresidue is synthesized. The oligonucleotide is then treated with anenzyme such as a UDG, which removes uracil, thereby producing an abasicresidue wherever uridine was present in the oligonucleotide.

In some embodiments of the invention the oligonucleotide probe comprisesa disaccharide nucleoside as described in Nauwelaerts, K., et al, Nuc.Acids. Res., 31(23), 2003. Following ligation, the extended duplex iscleaved using periodate (NaIO₄), followed by treatment with base (e.g.,NaOH) to remove the label, resulting in a free 3′ OH and P5-OPO₃H₂group. Depending on whether sequencing is performed in the 3′->5′ or5′→3′, it may be necessary or desirable to treat the extended duplexwith a polynucleotide kinase or phosphatase to generate an extendableterminus. Thus in certain methods of the invention an extendableterminus is generated by treatment with a polynucleotide kinase orphosphatase.

A polynucleotide comprising a disaccharide nucleoside is considered tocomprise an abasic residue. For example, a polynucleotide containing aribose residue inserted between the 3′OH of one nucleotide and the 5′phosphate group of the next nucleotide is considered to comprise anabasic residue.

Capping

In some cases, fewer than all probes with extendable termini participatein a successful ligation reaction in each cycle of extension, ligation,and cleavage. It will be appreciated that if such probes participated insucceeding cycles, the accuracy of each nucleotide identification stepwould progressively decline. Although the inventors have shown that useof extension probes containing phosphorothiolate linkages allowsligation with high efficiency, in certain embodiments of the invention acapping step is included to prevent those extendable termini that do notundergo ligation from participating in future cycles. When sequencing inthe 5′→3′ direction using extension probes containing a 3′-O—P—S-5′phosphorothiolate linkage, capping may be performed by extending theunligated extendable termini with a DNA polymerase and a non-extendablemoiety, e.g., a chain-terminating nucleotide such as a dideoxynucleotideor a nucleotide with a blocking moiety attached, e.g., following theligation or detection step. When sequencing in the 3′→5′ direction usingextension probes containing a 3′-S—P—O-5′ phosphorothiolate linkage,capping may be performed, e.g., by treating the template with aphosphatase, e.g., following ligation or detection. Other cappingmethods may also be used.

H. Sequencing Using Oligonucleotide Probe Families

In the sequencing methods described above, referred to collectively as“Methods A”, there is a direct and known correspondence between thelabel attached to any particular extension probe and the identity of oneor more nucleotides at the proximal terminus of the probe (i.e., theterminus that is ligated to the extendable probe terminus of theextended duplex. Therefore, identifying the label of a newly ligatedextension probe is sufficient to identify one or more nucleotides in thetemplate. The invention provides additional sequencing methods, referredto collectively as “Methods AB”, and also involving successive cycles ofextension, ligation, and, preferably, cleavage, that adopt a differentapproach to nucleotide identification.

The invention provide sequencing methods AB that use a collection of atleast two distinguishably labeled oligonucleotide probe families. Eachprobe family is assigned a name based on the label, e.g., “red”, “blue”,“yellow”, “green”. As in the methods described above, extension startsfrom a duplex formed by an initializing oligonucleotide and a template.The initializing oligonucleotide is extended by ligating anoligonucleotide probe to its end to form an extended duplex, which isthen repeatedly extended by successive cycles of ligation. The probe hasa non-extendable moiety in a terminal position (at the opposite end ofthe probe from the nucleotide that is ligated to the growing nucleicacid strand of the duplex) so that only a single extension of theextended duplex takes place in a single cycle. During each cycle, alabel on or associated with a successfully ligated probe is detected,and the non-extendable moiety is removed or modified to generate anextendable terminus. Detection of the label identifies the name of theprobe family to which the probe belongs.

Successive cycles of extension, ligation, and detection produce anordered series of label names. The labels correspond to the probefamilies to which successfully ligated probes that hybridize to thetemplate at successive positions belong. The probes have proximaltermini that are located opposite different nucleotides in the templatefollowing ligation. Thus there is a correspondence between the order ofprobe family names and the order of nucleotides in the template.

In embodiments of the invention in which the scissile linkage is locatedbetween the proximal nucleoside in the extension probe and the adjacentnucleoside, the ordered list of probe family names may be obtained bysuccessive cycles of extension, ligation, detection, and cleavage thatbegin from a single initializing oligonucleotide since the extendedoligonucleotide probe is extended by one nucleotide in each cycle. Ifthe scissile linkage is located between two of the other nucleosides,the ordered list of probe family names is assembled from resultsobtained from a plurality of sequencing reactions in which initializingoligonucleotides that hybridize to different positions within thebinding reaction are used, as described for sequencing methods A.

Knowing which probe family a newly ligated probe belongs to is not byitself sufficient to determine the identity of a nucleotide in thetemplate. Instead, determining the probe family name eliminates certaincombinations of nucleotides as possibilities for the sequence of atleast a portion of the probe but leaves at least two possibilities forthe identity of each nucleotide. Thus knowledge of the probe familyname, in the absence of additional information, leaves open least twopossibilities for the identity of the nucleotides in the template thatare located at opposite positions to the nucleotides in the newlyligated probe. Therefore any single cycle of extension, ligation,detection (and, optionally, cleavage) does not itself identify anynucleotide in the template. However, it does allow elimination of one ormore possible sequences for the template and thereby providesinformation about the sequence. In certain embodiments of the invention,with appropriate design of the probes and probe families as describedbelow, the sequence of the template can still be determined. In certainembodiments of the invention sequencing methods AB thus comprise twophases: a first phase in which an ordered list of probe family names isobtained, and a second phase in which the ordered list is decoded todetermine the sequence of the template.

Unless otherwise indicated, sequencing methods A and AB generally employsimilar methods for synthesizing probes, preparing templates, andperforming the steps of extension, ligation, cleavage, and detection.

Features of Oligonucleotide Extension Probes and Probe Families forSequencing Methods AB

Probe families for use in sequencing methods AB are characterized inthat each probe family comprises a plurality of labeled oligonucleotideprobes of different sequence and, at each position in the sequence, aprobe family comprises at least 2 probes having different bases at thatposition. Probes in each probe family comprise the same label.Preferably the probes comprise a scissile internucleoside linkage. Thescissile linkage can be located anywhere in the probe. Preferably theprobes have a moiety that is not extendable by ligase at one terminus.Preferably the probes are labeled at a position between the scissilelinkage and the moiety that is not extendable by ligase, such thatcleavage of the scissile linkage following ligation of a probe to anextendable probe terminus results in an unlabeled portion that isligated to the extendable probe terminus and a labeled portion that isno longer attached to the unlabeled portion.

The probes in each probe family preferably comprise at least jnucleosides X, wherein j is at least 2, and wherein each X is at least2-fold degenerate among the probes in the probe family. Probes in eachprobe family further comprise at least k nucleosides N, wherein k is atleast 2, and wherein N represents any nucleoside. In general, j+k isequal to or less than 100, typically less than or equal to 30.Nucleosides X can be located anywhere in the probe. Nucleosides X neednot be located at contiguous positions. Similarly nucleosides N need notbe located at contiguous positions. In other words, nucleosides X and Ncan be interspersed. Nevertheless, nucleosides X can be considered tohave a 5′→3′ sequence, with the understanding that the nucleosides neednot be contiguous. For example, nucleosides X in a probe of structureX_(A)NX_(G)NNX_(C)N would be considered to have the sequence AGC.Similarly, nucleosides N can be considered to have a sequence.

Nucleosides X can be identical or different but are not independentlyselected, i.e., the identity of each X is constrained by the identity ofone or more other nucleosides X in the probe. Thus in general onlycertain combinations of nucleosides X are present in any particularprobe and within the probes in any particular probe family. In otherwords, in each probe, the sequences of nucleosides X can only representa subset of all possible sequences of length j. Thus the identity of oneor more nucleotides in X limits the possible identities for one or moreof the other nucleosides.

Nucleosides N are preferably independently selected and can be A, G, C,or T (or, optionally, a degeneracy-reducing nucleoside). Preferably thesequence of nucleosides N represents all possible sequences of length k,except that one or more N may be a degeneracy-reducing nucleoside. Theprobes thus contain two portions, of which the portion consisting ofnucleosides N is referred to as the unconstrained portion and theportion consisting of nucleosides X is referred to as the constrainedportion. As described above, the portions need not be contiguousnucleosides. Probes that contain a constrained portion and anunconstrained portion are referred to herein as partially constrainedprobes. Preferably one or more nucleosides in the constrained portion isat the proximal end of the probes, i.e., at the end that contains thenucleoside that will be ligated to the extendable probe terminus, whichcan be either the 5′ or 3′ end of the oligonucleotide probe in differentembodiments of the invention.

Since the constrained portion of any oligonucleotide probe can only havecertain sequences, knowing the identity of one or more of thenucleosides in the constrained portion of a probe provides informationabout one or more of the other nucleosides. The information may or maynot be sufficient to precisely identify one or more of the othernucleosides, but it will be sufficient to eliminate one or morepossibilities for the identity of one or more of the other nucleosidesin the constrained portion. In certain preferred embodiments ofsequencing methods AB, knowing the identity of one nucleoside in theconstrained portion of a probe is sufficient to precisely identify eachof the other nucleosides in the constrained portion, i.e., to determinethe identity and order of the nucleosides that comprise the constrainedportion.

As in the sequencing methods described above, the most proximalnucleoside in an extension probe that is complementary to the templateis ligated to an extendable terminus of an initializing oligonucleotide(in the first cycle of extension, ligation, and detection) and to anextendable terminus of an extended oligonucleotide probe in subsequentcycles of extension, ligation, and detection. Detection determines thename of the probe family to which the newly ligated probe belongs. Sinceeach position in the constrained portion of the probe is at least 2-folddegenerate, the name of the probe family does not in itself identify anynucleotide in the constrained portion. However, since the sequence ofthe constrained portion is one of a subset of all possible sequences oflength j, identifying the probe family does eliminate certainpossibilities for the sequence of the constrained portion. Theconstrained portion of the probe constitutes its sequence determiningportion. Therefore, eliminating one or more possibilities for theidentity of one or more nucleosides in the constrained portion of theprobe by identifying the probe family to which it belongs eliminates oneor more possibilities for the identity of a nucleotide in the templateto which the extension probe hybridizes. In preferred embodiments of theinvention the partially constrained probes comprise a scissile linkagebetween any two nucleosides.

In certain embodiments the partially constrained probes have the generalstructure (X)_(j)(N)_(k), in which X represents a nucleoside, (X)_(j) isat least 2-fold degenerate at each position such that X can be any of atleast 2 nucleosides having different basepairing specificities, Nrepresents any nucleoside, j is at least 2, k is between 1 and 100, andat least one N or X other than the X at the probe terminus comprises adetectable moiety. Preferably (N)_(k) is independently 4-fold degenerateat each position so that, in each probe, (N)_(k) represents all possiblesequences of length k, except that one or more positions in (N)_(k) maybe occupied by a degeneracy-reducing nucleotide. Nucleosides in (X)_(j)can be identical or different but are not independently selected. Inother words, in each probe, (X)_(j) can only represent a subset of allpossible sequences of length j. Thus the identity of one or morenucleotides in (X)_(j) limits the possible identities for one or more ofthe other nucleosides. The probes thus contain two portions, of which(N)_(k) is the unconstrained portion and (X)_(j) is the constrainedportion.

In certain preferred embodiments of the invention the partiallyconstrained probes have the structure 5′-(X)_(j)(N)_(k)N_(B)*-3′ or3′-(X)_(j)(N)_(k)N_(B)*-5′, wherein N represents any nucleoside, N_(B)represents a moiety that is not extendable by ligase, * represents adetectable moiety, (X)_(j) is a constrained portion of the probe that isat least 2-fold degenerate at each position, nucleosides in (X)_(j) canbe identical or different but are not independently selected, at leastone internucleoside linkage is a scissile linkage, j is at least 2, andk is between 1 and 100, with the proviso that a detectable moiety may bepresent on any nucleoside N or X other than the X at the probe terminusinstead of, or in addition to, N_(B). The scissile linkage can bebetween two nucleosides in (X)_(j), between the most distal nucleotidein (X)_(j) and the most proximal nucleoside in (N)_(k), betweennucleosides within (N)_(k), or between the terminal nucleoside in(N)_(k) and N_(B). Preferably the scissile linkage is aphosphorothiolate linkage.

In yet more preferred embodiments of the invention the probes have thestructure 5′-(XY)(N)_(k)N_(B)*-3′ or 3′-(XY)(N)_(k)N_(B)*-5′, wherein Nrepresents any nucleoside, N_(B) represents a moiety that is notextendable by ligase, * represents a detectable moiety, XY is aconstrained portion of the probe in which X and Y represent nucleosidesthat are identical or different but are not independently selected, Xand Y are at least 2-fold degenerate, at least one internucleosidelinkage is a scissile linkage, and k is between 1 and 100, inclusive,with the proviso that a detectable moiety may be present on anynucleotide N or X other than the X at the probe terminus instead of, orin addition to, N_(B). Preferably the scissile linkage is aphosphorothiolate linkage. Probes having the structure5′-(XY)(N)_(k)N_(B)*-3′ are of use for sequencing in the 5′→3′direction. Probes having the structure 3′-(XY)(N)_(k)N_(B)*-5′ are ofuse for sequencing in the 3′→5′ direction.

The structure of certain preferred probes is represented in more detailas follows. For sequencing in the 5′→3′ direction, partially constrainedprobes having the structure5′-O—P—O—(X)_(j)(N)_(k)—O—P—S—(N)_(i)N_(B)*-3′ where N represents anynucleoside, N_(B) represents a moiety that is not extendable byligase, * represents a detectable moiety, (X)_(j) is a constrainedportion of the probe that is at least 2-fold degenerate at eachposition, nucleosides in (X)_(j) can be identical or different but arenot independently selected, j is at least 2, (k+i) is between 1 and 100,k is between 1 and 100, and i is between 0 and 99, with the proviso thata detectable moiety may be present on any nucleoside of (N)_(i), insteadof, or in addition to, N_(B). In certain embodiments of the invention(X)_(j), is (XY) in which X and Y are at least 2-fold degenerate andrepresent nucleotides that are identical or different but are notindependently selected. In certain embodiments of the invention i is 0.

Other preferred probes for sequencing in the 5′→3′ direction have thestructure 5′-O—P—O—(X)_(n)—O—P—S—(N)_(i)N_(B)*-3′ in which N representsany nucleoside, N_(B) represents a moiety that is not extendable byligase, * represents a detectable moiety, (X)_(j) is a constrainedportion of the probe that is at least 2-fold degenerate at eachposition, nucleotides in (X)_(j) can be identical or different but arenot independently selected, j is at least 2, and i is between 1 and 100,with the proviso that a detectable moiety may be present on anynucleoside of (N)_(i) instead of, or in addition to, N_(B). In certainembodiments of the invention (X)_(j) is (XY), in which positions X and Yare at least 2-fold degenerate and X and Y represent nucleosides thatare identical or different but are not independently selected. Yet otherpreferred probes for sequencing in the 5′→3′ direction have thestructure 5′-O—P—O—(X)_(j)—O—P—S—(X)_(k)(N)_(i)N_(B)*-3′ in which Nrepresents any nucleoside, N_(B) represents a moiety that is notextendable by ligase, * represents a detectable moiety,(X)_(j)—O—P—S—(X)_(k) is a constrained portion of the probe that is atleast 2-fold degenerate at each position, positions in(X)_(j)—O—P—S—X)_(k) are at least 2-fold degenerate and can be identicalor different but are not independently selected, j and k are both atleast 1 and (j+k) is at least 2 (e.g., 2, 3, 4, or 5), and i is between1 and 100, with the proviso that a detectable moiety may be present onany nucleoside of (N)_(i) instead of, or in addition to, N_(B). Incertain embodiments of the invention j and k are both 1.

For sequencing in the 3′→5′ direction, partially constrained probeshaving the structure 5′-N_(B)*(N)_(i)—S—P—O—(N)_(k)—O—P—O—(X)_(j)-3′where N represents any nucleoside, N_(B) represents a moiety that is notextendable by ligase, * represents a detectable moiety, (X)_(j) is aconstrained portion of the probe that is at least 2-fold degenerate ateach position, nucleosides in (X)_(j) can be identical or different butare not independently selected, j is at least 2, (k+i) is between 1 and100, k is between 1 and 100, and i is between 0 and 99, with the provisothat a detectable moiety may be present on any nucleoside of (N)_(i)instead of, or in addition to, N_(B). In certain embodiments of theinvention (X)_(j) is (XY) in which X and Y are at least 2-folddegenerate and represent nucleosides that are identical or different butare not independently selected. In certain embodiments of the inventioni is 0.

Other preferred probes for sequencing in the 3′→5′ direction have thestructure 5′-N_(B)*(N)_(i)—S—P—O—(X)_(j)-3′ in which N represents anynucleoside, N_(B) represents a moiety that is not extendable byligase, * represents a detectable moiety, (X)_(j) is a constrainedportion of the probe that is at least 2-fold degenerate at eachposition, nucleosides in (X)_(j) can be identical or different but arenot independently selected, j is at least 2, and i is between 1 and 100,with the proviso that a detectable moiety may be present on anynucleoside of (N)_(i) instead of, or in addition to, N_(B). In certainembodiments of the invention (X)_(j) is (XY) in which X and Y are atleast 2-fold degenerate and represent nucleosides that are identical ordifferent but are not independently selected. In certain embodiments ofthe invention j is between 2 and 5, e.g., 2, 3, 4, or 5, in any of thepartially constrained probes.

Yet other preferred probes for sequencing in the 3′→5′ direction havethe structure 5′-N_(B)*(N)_(i)—S—P—O—(X)_(k)—O—P—O—(X)_(j)-3′ where Nrepresents any nucleoside, N_(B) represents a moiety that is notextendable by ligase, * represents a detectable moiety,—(X)_(k)—O—P—O—(X)_(j) is a constrained portion of the probe that is atleast 2-fold degenerate at each position, nucleosides in—(X)_(k)—O—P—O—(X)_(j) can be identical or different but are notindependently selected, j and k are both at least 1 and (j+k) is atleast 2 (e.g., 2, 3, 4, or 5), i is between 1 and 100, with the provisothat a detectable moiety may be present on any nucleoside of (N)_(i)instead of, or in addition to, N_(B). In certain embodiments j=1 andk=1.

In embodiments of the invention in which the scissile linkage is locatedbetween the most proximal nucleoside in (X)_(j) and the next mostproximal nucleoside in (X)_(j), the ordered list of probe family namesmay be obtained by successive cycles of extension, ligation, detection,and cleavage that begin from a single initializing oligonucleotide sincethe extended oligonucleotide probe is extended by one nucleotide in eachcycle. In embodiments of the invention in which the scissile linkage islocated between two of the other nucleosides, the ordered list of probefamily names is assembled from results obtained from a plurality ofsequencing reactions in which initializing oligonucleotides thathybridize to different positions within the binding reaction are used,as described for sequencing methods A.

It will be understood that probes having any of a large number ofstructures other than those described above can be employed insequencing methods AB. For example, probes can have structures such asXNY(N)_(k) in which the constrained nucleosides X and Y are notadjacent, or XIY(N)_(k) where I is a universal base. (N)_(k)X(N)_(l),(N)_(i)X(N)_(j)Y(N)_(k)Z(N)_(l), (N)_(i)X(N)_(j)YIZ(N)_(l), and(N)_(i)X(N)_(j)Y(N)_(k)Z(I)_(l) represent additional possibilities. Asin the probes described above, these probes comprise a scissile linkage,a detectable moiety, and a moiety at one terminus that is not extendableby ligase. Preferably the probes do not comprise a detectable moietyattached to the nucleotide at the opposite end of the probe from themoiety that is not extendable by ligase. Probe families comprisingprobes having any of these structures, or others, satisfy the criterionthat each probe family comprises a plurality of labeled probes ofdifferent sequence and, at each position in the sequence, a probe familycomprises at least 2 probes having different bases at that position.Preferably the total number of nucleosides in each probe is 100 or less,e.g., 30 or less.

Encoding Oligonucleotide Extension Probe Families. The inventivesequencing method makes use of encoded probe families. An “encoding”refers to a scheme that associates a particular label with a probecomprising a portion that has one of a defined set of sequences, suchthat probes comprising a portion that has a sequence that is a member ofthe defined set of sequences are labeled with the label. In general, anencoding associates each of a plurality of distinguishable labels withone or more probes, such that each distinguishable label is associatedwith a different group of probes, and each probe is labeled by only asingle label (which can comprise a combination of detectable moieties).Preferably the probes in each group of probes each comprise a portionthat has a sequence that is a member of the same defined set ofsequences. The portion may be a single nucleoside or may be multiplenucleosides in length, e.g., 2, 3, 4, 5, or more nucleosides in length.The length of the portion may constitute only a small fraction of theentire length of the probe or may constitute up to the entire probe. Thedefined set of sequences may contain only a single sequence or maycontain any number of different sequences, depending on the length ofthe portion. For example, if the portion is a single nucleoside, thedefined set of sequences could have at most 4 elements (A, G, C, T). Ifthe portion is two nucleosides in length, the defined set of sequencescould have up to 16 elements (AA, AG, AC, AT, GA, GG, GC, GT, CA, CG,CC, CT, TA, TG, TC, TT). In general, the defined set of sequences willcontain fewer elements than the total number of possible sequences, andan encoding will employ more than one defined set of sequences.

Sequencing methods A described herein generally make use of a set ofprobes having a simple encoding in which there is a directcorrespondence between the proximal nucleoside in the probe (i.e., thenucleoside that is ligated to the extendable probe terminus) and theidentity of the label. The proximal nucleoside is complementary to thenucleotide with which it hybridizes in the template, so the identity ofthe proximal nucleoside in a newly ligated probe determines the identityof the nucleotide in the template that is located at the oppositeposition in the extended duplex. In a general sense, probes of use inthe other sequencing methods described herein have the structureX(N)_(k), in which X is the proximal nucleoside, and each nucleoside Nis 4-fold degenerate, such that all possible sequences of length k arerepresented in the pool of oligonucleotide probe molecules thatconstitutes the probe. Thus, for example, some oligonucleotide probemolecules will contain A at position k=1, others will contain G atposition k=1, others will contain C at position k=1, others will containT at position k=1, and similarly for other positions k, where thenucleoside adjacent to X in (N)_(k) is considered to occupy positionk=1; the next nucleoside in (N)_(k) is considered to occupy positionk=2, etc. However, in any given oligonucleotide probe, X represents onlya single base pairing specificity, which typically corresponds to aparticular nucleoside identity, e.g., A, G, C, or T. Thus X is typicallyuniformly A, G, C, or T in the pool of probe molecules that constitute aparticular probe. FIG. 2 shows a suitable encoding for probes having thestructure X(N)_(k). According to this encoding, probes having X=C areassigned the label “red”; probes having X=A are assigned the label“yellow”; probes having X=G are assigned the label “green”; and probeshaving X=T are assigned the label “blue”. Thus there is a one-to-onecorrespondence between the sequence determining portion of the probe andits label.

It will be appreciated that the above approach in which the identity ofthe label of a newly ligated extension probe corresponds to the identityof the most proximal nucleoside in the extension probe may be broadenedto encompass encodings in which the identity of the label correspondsnot to the identity of only the most proximal nucleoside in theextension probe but rather to the sequence of the most proximal 2 ormore nucleosides in the extension probe, so that the identity ofmultiple nucleotides in the template can be determined in a single cycleof extension, ligation, and detection (typically followed by cleavage).However, such encodings would still associate a label with a singlesequence in the oligonucleotide extension probe so that the identity ofthe oppositely located complementary nucleotides in the template couldbe identified. For example, as described above, in order to identify twonucleotides in a single cycle, 16 different oligonucleotide probes, eachwith a corresponding label (i.e., 16 distinguishable labels) would beneeded.

Sequencing method AB employs an alternative approach to associatinglabels with probes. Rather than a one-to-one correspondence between theidentity of the label and the sequence of the sequence determiningportion of the probe, the same label is assigned to multiple probeshaving different sequence determining portions. The probes are partiallyconstrained, and the constrained portion of the probe is its sequencedetermining portion. Thus the same label is assigned to a plurality ofdifferent probes, each having a constrained portion with a differentsequence, wherein the sequence is one of a defined set of sequences. Asmentioned above, probes comprising the same label constitute a “probefamily”. The method employs a plurality of such probe families, eachcomprising a plurality of probes having a constrained portion with adifferent sequence, wherein the sequence is one of a defined set ofsequences.

A plurality of probe families is referred to as a “collection” of probefamilies. Probes in each probe family in a collection of probe familiesare labeled with a label that is distinguishable from labels used tolabel other probe families in the collection. Each probe familypreferably has its own defined set of sequences. Preferably theconstrained portions of the probes in each probe family are the samelength, and preferably the constrained portions of probe families in acollection of probe families are of the same length. Preferably thecombination of sets of defined sequences for probe families in acollection of probe families includes all possible sequences of thelength of the constrained portion. Preferably a collection of probefamilies comprises or consists of 4 distinguishably labeled probefamilies. Preferably the constrained portion of the probes is 2nucleosides in length.

A wide variety of differently encoded collections of distinguishablylabeled probe families will satisfy the above criteria and may be usedto practice the inventive method. However, certain collections of probefamilies are preferred. An exemplary encoding for a preferred collectionof 4 distinguishably labeled probe families comprising partiallyconstrained probes is shown in FIG. 25A. As depicted in FIG. 25A, theconstrained portion consists of the 2 most 3′ nucleosides in the probe.The probe families are labeled “red”, “yellow”, “green”, and “blue”.Probes in each probe family comprise a constrained portion whosesequence is one of a defined set of sequences, the defined set beingdifferent for each probe family. For example, beginning at the 3′ end ofeach sequence, which is considered to be the proximal end of the probe,the defined set of sequences for the “red” probe family is {CT, AG, GA,TC}; the defined set of sequences for the “yellow” probe family is {CC,AT, GG, TA}; the defined set of sequences for the “green” probe familyis {CA, AC, GT, TG}; the defined set of sequences for the “blue” probefamily is {CG, AA, GC, TT}. Each defined set does not contain any memberthat is present in one of the other sets, a characteristic that ispreferred. In addition, the combination of sets of defined sequences forprobe families in a collection of probe families includes all possiblesequences of length 2, i.e., all possible dinucleosides. Anothercharacteristic of this collection of probe families, which is preferredbut not required, is that each position in the constrained portion ofthe probes is 4-fold degenerate, i.e., it can be occupied by either A,G, C, or T. Another characteristic of this collection of probe families,which is preferred but not required, is that within each set of definedsequences only a single sequence has any specific nucleoside at anyposition, e.g., at the most proximal position or at any of otherpositions. It is particularly preferred, but not required, that withineach set of defined sequences only a single sequence has any specificnucleoside at position 2 or higher within the constrained portion,considering the most proximal nucleoside to be at position 1. Forexample, in the defined set of sequences for the Red probe family, onlyone sequence has T at position 2; only one sequence has G at position 2;only one sequence has A at position 2; only one sequence has C atposition 2.

Given any particular encoding such as that depicted in FIG. 25A, knowingthe identity of one or more nucleosides in the constrained portion of aprobe in one of the probe families provides information about the othernucleotides in the constrained portion of that probe. In the mostgeneral sense, knowing the identity of one or more nucleosides in theconstrained portion of a probe in a probe family provides sufficientinformation to eliminate one or more possible identities for anucleoside at one of the other positions, because the defined set ofsequences for that probe family will not contain a sequence having anucleoside with that identity at that position. Typically knowing theidentity of one or more nucleosides in the constrained portion of aprobe in a probe family provides sufficient information to eliminate oneor more possible identities for a plurality of nucleosides, e.g., eachof the other nucleosides. For preferred encodings, knowing the identityof one or more nucleosides in the constrained portion of a probe in theprobe family eliminates all but one possibility for each of the othernucleosides in the probe. For example, in the case of the encoded probefamilies shown in FIG. 25A, if it is known that a probe is a member ofthe red family, and if it is also known that the most proximalnucleoside is C, then the adjacent nucleoside must be T. Similarly, ifit is known that a probe is a member of the green family, and if it isalso known that the most proximal nucleoside is G, then the adjacentnucleoside must be T. Thus knowing the identity of one nucleoside in theconstrained portion is sufficient to eliminate all possibilities for theother nucleoside except one, so the identity of the other nucleoside iscompletely specified. Yet without knowing the identity of at least onenucleoside in the constrained portion of a probe it is not possible togain any information at all about the identity of any specificnucleoside in the probe based only on knowing the name of the probefamily to which it belongs since the nucleoside at each position of theconstrained portion could be A, G, C, or T. FIG. 25B shows a preferredcollection of probe families (upper panel) and a cycle of ligation,detection, and cleavage (lower panel) using sequencing methods AB.

The inventors have designed 24 collections of probe families containingconstrained portions that are 2 nucleosides in length and that have theadvantageous features of the collection of probe families depicted inFIG. 25A. These probe families are maximally informative in that knowingthe name of the probe family to which a probe belongs, and knowing theidentity of one nucleoside in the probe, is sufficient to preciselyidentify the other nucleoside in the constrained portion. This is thecase for all probes, and for all nucleosides in each constrainedportion. The encoding schemes for each of the 24 preferred collectionsof probe families are shown in Table 1. Table 1 assigns an encoding IDranging from 1 to 24 to each collection of probe families. Each encodingdefines the constrained portions of a collection of preferred probefamilies of general structure (XY)N_(k) for use in sequencing methodsAB, and thereby defines the collection itself. In Table 1 a value of 1in the column under an encoding ID indicates that, according to thatencoding, a probe comprising nucleosides X and Y as indicated in thefirst and second columns, respectively, is assigned to the first probefamily; (ii) a value of 2 in the column under an encoding ID indicatesthat, according to that encoding, a probe comprising nucleosides X and Yas indicated in the first and second columns, respectively, is assignedto the second probe family; (iii) a value of 3 in the column under anencoding ID indicates that, according to that encoding, a probecomprising nucleosides X and Y as indicated in the first and secondcolumns, respectively, is assigned to the third probe family; and (iv) avalue of 4 in the column under an encoding ID indicates that, accordingto that encoding, a probe comprising nucleosides X and Y as indicated inthe first and second columns, respectively, is assigned to the fourthprobe family. The values 1, 2, 3, and 4, each represent a label. Forexample, encoding 9 defines the collection of probe families depicted inFIG. 25A, in which 1 represents blue, 2 represents green, 3 representsred, and 4 represents yellow. It will be appreciated that the assignmentof values to labels is arbitrary, e.g., 1 could equally well representgreen, red, or yellow. Changing the association between values 1, 2, 3,and 4, and the labels would not change the set of probes in each probefamilies but would merely associate a different label with each probefamily.

TABLE 1 Oligonucleotide Probe Family Encodings Encoding ID X Y 1 2 3 4 56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A A 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C A 2 4 3 2 2 4 3 2 2 3 4 3 2 3 3 4 2 34 4 2 4 3 4 G A 4 3 2 3 3 2 4 4 3 2 2 4 4 2 4 3 4 2 3 2 3 2 4 3 T A 3 24 4 4 3 2 3 4 4 3 2 3 4 2 2 3 4 2 3 4 3 2 2 A C 2 2 2 2 2 2 2 2 2 2 2 22 2 2 2 2 2 2 2 2 2 2 2 C C 1 1 1 1 1 1 1 1 4 4 3 4 4 4 4 3 3 4 3 3 3 34 3 G C 3 4 4 4 4 3 3 3 1 1 1 1 3 3 3 4 1 1 1 1 4 4 3 4 T C 4 3 3 3 3 44 4 3 3 4 3 1 1 1 1 4 3 4 4 1 1 1 1 A G 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 33 3 3 3 3 3 3 3 C G 4 2 4 4 4 2 4 4 1 1 1 1 1 1 1 1 4 2 2 2 4 2 2 2 G G1 1 1 1 2 4 2 2 4 4 4 2 2 4 2 2 2 4 4 4 1 1 1 1 T G 2 4 2 2 1 1 1 1 2 22 4 4 2 4 4 1 1 1 1 2 4 4 4 A T 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 4 4 4 C T 3 3 2 3 3 3 2 3 3 2 2 2 3 2 2 2 1 1 1 1 1 1 1 1 G T 2 2 3 21 1 1 1 2 3 3 3 1 1 3 1 3 3 2 3 2 3 2 2 T T 1 1 1 1 2 2 3 2 1 1 1 1 2 31 3 2 2 3 2 3 2 3 3

To further illustrate the use of Table 1 to define the collections ofpreferred probe families, consider encoding 17. According to thisencoding, probes having constrained portions AA, GC, TG, and CT areassigned to label 1 (e.g., red); probes having constrained portions CA,AC, GG, and TT are assigned to label 2 (e.g., yellow); probes havingconstrained portions TA, CC, AG, and GT are assigned to label 3 (e.g.,green); and probes having constrained portions GA, TC, CG, and AT areassigned to label 4 (e.g., blue). The resulting collection of probefamilies is depicted in FIG. 26.

FIGS. 27A-27C represent an alternate method to schematically define the24 preferred collections of probe families. The method makes use ofdiagrams such as that in FIG. 27A. The first column in such a diagramrepresents the first base. Each label is attached to four different basesequences, each of which is given by juxtaposing the base from the firstcolumn with the base from the chosen label's column. For example, ifthere is an A in the column with the heading “First base”, then a probewith constrained portion having sequence AA is assigned to probe family1 (label 1); a probe with constrained portion having sequence AC isassigned to probe family 2 (label 2); a probe with constrained portionhaving sequence AG is assigned to probe family 3 (label 3); and a probewith constrained portion having sequence AT is assigned to probe family4 (label 4). Assignments to probe families are made in a similar mannerfor probes with constrained portions beginning with C, G, or T. Thus adiagram filled in with bases as shown in FIG. 27A translates to theencoding shown in FIG. 27B, in which probes having constrained portionsin the set {AA, CC, GG, TT} are assigned to probe family 1; probeshaving constrained portions in the set {AC, CA, GT, TG} are assigned toprobe family 2; probes having constrained portions in the set {AG, CT,GC, TA} are assigned to probe family 3; and probes having constrainedportions in the set {AT, CG, GA, TC} are assigned to probe family 4.FIG. 27C shows diagrams that may be inserted in place of the shadedportion of the diagram in FIG. 27A in order to generate each of the 24preferred collections of probe families. Methods of using the preferredcollections of probe families in sequencing methods AB are describedfurther below.

The 24 collections of encoded probe families defined by Table 1represent only the preferred embodiments of collections of probefamilies for use in sequencing methods AB. A wide variety of otherencoding schemes, probe families, and probe structures can be used thatemploy the same basic principle, in which knowing a probe family name,together with knowledge of the identity of one or more nucleosides in aconstrained portion, provides information about one or more othernucleosides. As compared with a preferred collection of probe families,the less preferred collections of probe families are generally lesspreferred because: (i) at least with respect to some probes, the amountof information afforded by knowing a probe family name and a nucleosideidentity is less; or (ii) at least with respect to some probes, theamount of information afforded by knowing a probe family name is more.

In general, less preferred collections of probe families may be used toperform sequencing methods AB in a similar manner to the way in whichpreferred collections of probe families are used. However, the stepsneeded for decoding may differ. For example, in some situationscomparing candidate sequences with each other may be sufficient todetermine at least a portion of a sequence.

An example of a less preferred collection of probe families in which theprobes comprise constrained portions that are 2 nucleosides in length isshown in FIG. 28. According to this encoding, probes having constrainedportions in the set {AA, AC, GA, GC} are assigned to probe family 1;probes having constrained portions in the set {CA, CC, TA, TC} areassigned to probe family 2; probes having constrained portions in theset {AG, AT, GG, GT} are assigned to probe family 3; and probes havingconstrained portions in the set {CG, CT, TG, TT} are assigned to probefamily 4. In this collection of probe families, knowing the name of aprobe family eliminates certain possibilities for the identity of anucleotide in the template that is located opposite the proximalnucleoside in a newly ligated extension probe whose label was detectedto determine the name of the probe family. For example, if the probefamily name is 1, then the proximal nucleoside in a newly ligatedextension probe must be A or G, so the complementary nucleotide in thetemplate must be T or C. Since there are at least two possibilities ateach position in the constrained portion, the nucleotide cannot beprecisely identified, but information sufficient to rule out somepossibilities is obtained from the single cycle, in contrast to thesituation when preferred collections of probe families are employed.

In certain embodiments of the invention partially constrained probes inwhich the constrained portion is 3 nucleosides in length are used. Inorder to contain probes whose constrained portions include all possiblesequences of length 3, as is preferred, the collection of probe familiesshould comprise 4³=64 different probes. FIG. 29A shows a diagram thatcan be used to generate constrained portions for a collection of probefamilies that comprises probes with a constrained portion 3 nucleosideslong (trinucleosides). The figure shows 4 sets of rows indicated A, G,C, and T, and 4 columns with probe family names 1, 2, 3, and 4. Each setof 4 rows is opposite a box with a nucleoside identity inside. Todetermine a probe family for a trinucleoside, the box containing thelast nucleoside in the trinucleoside is first selected. Within the fourrows adjacent to that box, the row labeled with the letter identifyingthe first nucleoside in the trinucleoside is selected. Within that row,the column containing the second nucleoside of the trinucleoside isselected. The trinucleoside is assigned to the probe family indicated atthe top of the column. For example, the following procedure is followedto assign the trinucleoside “TCG” to a probe family: Since the lastnucleoside is a “G”, attention is confined to the set of 4 rows locatedopposite the box containing “G”, i.e., the third set of rows. Since thefirst nucleoside is “T”, consideration is further limited to the lastrow in the set of 4. The probe family assignment is determined by theheading of the column that contains middle nucleoside. Since the middlenucleoside is “C”, the trinucleoside is assigned to probe family 1. Asimilar process yields the following probe family assignments: AAA=1;ATA=2; AGA=3; GTA=4; GAG=1; TGG=2, etc. The process continues until allpossible trinucleosides have been assigned to a probe family.

FIG. 29B shows a procedure for constructing additional constrainedportions for a collection of probe families that comprises probes with aconstrained portion 3 nucleosides long. The procedure is used toconstruct such a collection from each of the 24 preferred collections ofprobe families described above, in which constrained portions are 2nucleosides in length and the collection contains 4 probe families. Anexemplary diagram representing a preferred collection of probe familiesis shown in the upper portion of the figure. The columns of this diagrammap directly into the columns of the lower portion of the figure inaccordance with the color assigned to each column in the upper diagram.Thus the columns in the upper diagram are blue, green, yellow, and red,moving from left to right. The entries under column 1 in the lowerdiagram are blue, green, yellow, and red, moving from top to bottom,with each set of 4 nucleosides corresponding to a column in the upperdiagram. Columns 2, 3, and 4 in the lower diagram are generated byprogressively moving each set of 4 nucleosides in column 1 downwards.

It will be appreciated that a “probe family” can be considered to be asingle “super-probe” comprising a plurality of different probes, eachwith the same label. In this case, the probe molecules that constitutethe probe will generally not be a population of substantially identicalmolecules across any portion of the probe. Use of the term “probefamily” is not intended to have any limiting effect but is used forconvenience to describe the characteristics of probes that wouldconstitute such a “super-probe”.

Decoding

As described above, successive cycles of extension, ligation, detection,and cleavage using a collection of probe families comprising at leasttwo distinguishably labeled probe families yields an ordered list ofprobe family names either from a single sequencing reaction or fromassembling probe family names determined in multiple sequencingreactions that initiate from different sites in the template into anordered list. The number of cycles performed should be approximatelyequivalent to the length of sequence desired. The ordered list containsa substantial amount of information but not in a form that willimmediately yield the sequence of interest. Further step(s), at leastone of which involves gathering at least one item of additionalinformation about the sequence, must be performed in order to obtain asequence that is most likely to represent the sequence of interest. Thesequence that is most likely to represent the sequence of interest isreferred to herein as the “correct” sequence, and the process ofextracting the correct sequence from the ordered list of probe familiesis referred to as “decoding”. It will be appreciated that elements in an“ordered list” as described above could be rearranged either duringgeneration of the list or thereafter, provided that the informationcontent, including the correspondence between elements in the list andnucleotides in the template, is retained, and provided that therearrangement, fragmentation, and/or permutation is appropriately takeninto consideration during the decoding process (discussed below). Theterm “ordered list” is thus intended to encompass rearranged,fragmented, and/or permuted versions of an ordered list generated asdescribed above, provided that such rearranged, fragmented, and/orpermuted versions include substantially the same information content.

The ordered list can be decoded using a variety of approaches. Some ofthese approaches involve generating a set of at least one candidatesequence from the ordered list of probe family names. The set ofcandidate sequences may provide sufficient information to achieve anobjective. In preferred embodiments one or more additional steps areperformed to select the sequence that is most likely to represent thesequence of interest from among the candidate sequences or from a set ofsequences with which the candidate sequence is compared. For example, inone approach at least a portion of at least one candidate sequence iscompared with at least one other sequence. The correct sequence isselected based on the comparison. In certain embodiments of theinvention, decoding involves repeating the method and obtaining a secondordered list of probe family names using a collection of probe familiesthat is encoded differently from the original collection of probefamilies. Information from the second ordered list of probe families isused to determine the correct sequence. In some embodiments informationobtained from as little as one cycle of extension, ligation, anddetection using the alternately encoded collection of probe families issufficient to allow selection of the correct sequence. In other words,the first probe family identified using the alternately encoded probefamily provides sufficient information to determine which candidatesequence is correct.

Other decoding approaches involve specifically identifying at least onenucleotide in the template by any available sequencing method, e.g., asingle cycle of sequencing method A. Information about the one or morenucleotide(s) is used as a “key” to decode the ordered list of probefamily names. Alternately, the portion of the template that is sequencedmay comprise a region of known sequence in addition to a region whosesequence is unknown. If sequencing methods AB are applied to a portionof the template that includes both unknown sequence and at least onenucleotide of known sequence, the known sequence can be used as a “key”to decode the ordered list of probe family names. The following sectiondescribes the process of generating candidate sequences. Subsequentsections describe using the candidate sequences to select the correctsequence by comparing with known sequences, by comparing with a secondset of candidate sequences, and by utilizing a known nucleotideidentity.

Generating Candidate Sequences

It will be appreciated that the region of the template to be sequencedis complementary to the extended duplex that is produced by successivecycles of extension, ligation, and cleavage. Therefore, generating acandidate sequence for the extended duplex is equivalent to generating acandidate sequence for the region of the template to be sequenced. Inpractice, one could generate candidate sequences for the region of thetemplate to be sequenced, or one could generate candidate sequences forthe extended duplex and take their complement to determine candidatesequences for the region of the template to be sequenced. The latterapproach is described here. To generate a candidate sequence from a listof probe family names, the first member of the list of probe families isconsidered. The set of constrained portions associated with that probefamily limits the possibilities for the initial nucleotides in thesequence, out to a length equivalent to the length of the constrainedportion. For example, if the constrained portion is a dinucleotide, thenthe possible sequences for the first dinucleotide in the extended duplexare limited to those constrained portions that occur in probes that fallwithin that probe family (and thus the possible sequences for the firstdinucleotide in the region of the template to be sequenced are limitedto those combinations that are complementary to the constrained portionsthat occur in probes that fall within that probe family). Thepossibilities for the first dinucleotide are recorded, typically by acomputer. Similarly, the possible sequences for the second dinucleotidein the extended duplex (i.e., the dinucleotide that is one nucleotideoffset from the first dinucleotide) are limited to those constrainedportions that occur in probes that fall within the second probe family(and therefore, the possible sequences for the second dinucleotide inthe template, i.e., the dinucleotide that is one nucleotide offset fromthe first dinucleotide are limited to those combinations that arecomplementary to the constrained portions that occur in probes that fallwithin the second probe family). The possible sequences for the seconddinucleotide are also recorded. Possibilities for succeedingdinucleotides are likewise recorded until possibilities have beenrecorded for dinucleotides that correspond to the desired length of thesequence to be determined or there are no more probe families in thelist.

A representative example of the process of recording possibilities isdepicted in FIG. 30, in which it is assumed that a list of probe familynames has been generated using the probe family collection shown in FIG.25A. The leftmost column of FIG. 30 shows the list of probe families inorder from top to bottom: Yellow, Green, Red, Blue. The sequencepossibilities for the dinucleotide corresponding to each probe family inthe list are shown on the right side of the figure. Nucleotide positionsare indicated above the sequence possibilities. The sequence begins atposition 1, so the first dinucleotide occupies positions 1 and 2; thesecond dinucleotide occupies positions 2 and 3, etc. For the Yellowprobe family, the possibilities are CC, AT, GG, and TA, as shown in FIG.30. For the Green probe family, the possibilities are CA, AC, GT, andTG, etc. The process of recording the possible sequences of eachdinucleotide is continued until a desired sequence length has beenreached.

After the sets of possibilities are generated, a first assumption ismade about the identity of the first nucleotide in the candidatesequence, which is assumed to be at the 5′ position of the sequence,indicated as position 1 in FIG. 30. The first assumption can be that thenucleotide is A, that the nucleotide is G, that the nucleotide is C, orthat the nucleotide is T.

It will be observed that the possible sequences for each dinucleotideare limited by the possible sequences of the adjacent dinucleotides,since adjacent dinucleotides overlap, i.e., the second nucleotide of thefirst dinucleotide is also the first nucleotide of the seconddinucleotide. For example, if the first nucleotide is assumed to be C,then the first dinucleotide must be CC. If the first dinucleotide is CC,then the second dinucleotide must have a C at its first position. Sincethe only possible sequence for the second dinucleotide that has a C atits first position is CA, it is evident that the second dinucleotidemust be CA. Therefore the sequence of the first 3 nucleotides must beCCA. Similarly, the possible sequences for the third dinucleotide arelimited by the possible sequences of the second dinucleotide. If thesecond dinucleotide is CA, then the third dinucleotide must be AG sincethat is the only possibility that has A at its first position. Thus thesequence of the first 4 nucleotides must be CCAG. Continuing thisprocess results in a sequence of 5′-CCAGC-3′ for the first 5nucleotides. CCAGC is thus the first candidate sequence.

A second candidate sequence is generated by assuming that the firstnucleotide is A. This assumption yields AT for the first dinucleotide.TG is the only possible sequence for the second dinucleotide that isconsistent with a sequence of AT for the first dinucleotide. GA is theonly possible sequence for the third dinucleotide that is consistentwith a sequence of TG for the second dinucleotide. AA is the onlypossible sequence for the fourth dinucleotide that is consistent with asequence of GA for the third dinucleotide. Assembling thesedinucleotides into a full length candidate sequence yields ATGAA.Similarly, an assumption that the first nucleotide is a G yields thecandidate sequence GGTCG, and an assumption that the first nucleotide isa T yields the candidate sequence TACTT. Thus 4 candidate sequences aregenerated, each beginning with a different nucleotide assumed to be thefirst nucleotide in the sequence.

There is no requirement that the assumption must be made about the firstnucleotide rather than one of the other nucleotides. For example, anassumption could equally well have been made about the identity of thefourth nucleotide, in which case the candidate sequences would have beengenerated by moving “backwards” along the template (i.e., in a 3′→5′direction). For example, assuming that the fourth nucleotide is T meansthat the fourth dinucleotide must be TT; the third dinucleotide must beCT; the second dinucleotide must be AC; and the first dinucleotide mustbe CC. (Nucleotides are written in the 5′→3′ orientation although theiridentities are generated by moving from 3′→5′ in the sequence.)Alternately, an assumption can be made about any nucleotide in themiddle of the sequence, and dinucleotide identities generated by movingboth in the 5′→3′ and the 3′→5 directions. It will be appreciated thatin the absence of an assumption about one of the nucleotides, theidentity of each nucleotide remains completely undetermined since eachposition could be occupied by A, G, C, or T.

When using preferred collections of probe families, assuming theidentity of any single nucleotide (e.g., the first nucleotide) generatesone and only one candidate sequence. However, when less preferredcollections of probe families are used it may be necessary to assume anidentity for more than one nucleotide, i.e., assuming an identity for afirst nucleotide does not entirely specify the rest of the sequence. Forexample, a less preferred collection of probe families may include afamily with members whose defined sequences are AA and AC. In such acase, assuming that the first nucleotide is A leaves two possibilitiesfor the second nucleotide. Sequencing using less preferred collectionsof probe families is discussed further below. It will be appreciatedthat if the constrained portions consist of noncontiguous nucleotides,the approach described above can still be used with minor modifications.

Sequence Identification by Comparing Candidate Sequences with KnownSequences

Generally if the candidate sequences of the extended duplexes weredetermined, as described above, corresponding candidate sequences forthe region of the template to be sequenced are obtained by taking theircomplements. In some instances, the candidate sequences themselves willprovide enough information to achieve an objective. For example, if thepurpose of sequencing is simply to rule out certain sequencepossibilities, then comparing the candidate sequences with thosepossibilities would be sufficient. The candidate sequences shown in FIG.30 would allow a determination that the region being sequenced was notpart of a polyA tail, for example. A longer sequence could confirm thatthe region being sequenced was not part of a vector.

In many instances it will be desirable to explicitly determine thecorrect sequence. According to a preferred embodiment of the inventionthe correct sequence is identified by comparing the candidate sequencesfor the region of the template to be sequenced with a set of knownsequences. The set of known sequences may, for example, be a set ofsequences for a particular organism of interest. For example, if humanDNA is being sequenced, then the candidate sequences can be comparedwith the Human Draft Genome Sequence. See the National Center forBiotechnology Information (NCBI) web site for a guide to publiclyavailable human genome sequence resources. As another example, ifnucleic acid derived from an infectious agent (e.g., a bacterium orvirus isolated from a subject) is being sequenced, a database containingsequences of variant strains of that bacterium or virus can be searched.Many such organism-specific databases, containing either complete orpartial sequences, are known in the art, and more will become availableas sequencing efforts accelerate. Some representative examples includedatabases for the mouse, human immunodeficiency virus, malaria speciesPlasmodium falciparum, etc. Of course it is not necessary to use anorganism-specific set of sequences. A database such as GenBank, whichcontains sequences from a wide variety of organisms and viruses, can besearched. The database need not even contain any sequences from theorganism or virus from which the template was derived. In general, thesequences can be genomic sequences, cDNA sequences, ESTs, etc. Multiplesequences can be searched.

Simply performing the search may be sufficient to achieve an objective.For example, if viral nucleic acid is isolated from a patient, comparingthe candidate sequences with a set of known sequences of that virus candetermine that the viral nucleic acid either does or does not containsequences from that virus, even if the matching sequence is neverexamined. The existence of a match would confirm that the patient isinfected with the virus, while lack of a match would indicate that thepatient is not infected with the virus.

In certain embodiments the set of known sequences contains a narrowerrange of sequences, which may be specifically tailored to the purposefor which the sequencing is performed. Thus information about thenucleic acid being sequenced may be used to select the set of knownsequences. For example, if it is known that the template representssequence of a particular gene, the known sequences may representdifferent alleles of a gene, mutant and wild type sequences at a givenlocus of interest, etc. It may only be necessary to compare thecandidate sequences with a single known sequence to determine which ofthe candidate sequences is correct. For example, in certain embodimentsof the invention the template is obtained by amplifying DNA thatcontains a region of interest (e.g., using primers that flank the regionof interest). The region of interest may encompass a site at whichmutations or polymorphisms may exist, e.g., mutations or polymorphismsthat are associated with a particular disease. If it is known that thetemplate represents a sequence from a particular region of interest,then the candidate sequences need only be compared with a singlereference sequence for that region, e.g., a wild type or mutant form ofthe sequence. In other words, if part or all of the sequence of thetemplate is known, it may not be necessary to perform a comparison witha plurality of known sequences. Instead, a candidate sequence thatcomprises all or part of the known sequence is selected as correct. Forexample, mutations in the BRCA1 and BRCA2 genes are known to beassociated with an increased risk of breast cancer, and there issignificant interest in determining whether subjects carry suchmutations. If it is known that the template comprises sequence from theBRCA1 gene, e.g., if primers flanking a region of interest thatencompasses a portion of the gene were used to produce a clonalpopulation of templates, then the candidate sequences need only becompared against the wild type or mutant BRCA1 sequence to determine thecorrect sequence.

In the more general case, comparing the candidate sequences with the setof known sequences will identify any known sequences that are similar toany of the candidate sequences. Provided that the candidate sequencesare of sufficient length, the likelihood that a database will containsequences that is identical to or closely resemble more than one of thecandidate sequences are very small. In other words, if the candidatesequences are long enough, it is unlikely that more than one of themwill be represented in the set of known sequences. The candidatesequences are compared with any sequences that are considered to be a“match”. It will typically be desirable to set a threshold for thedegree of identity required to establish that a match exists. Forexample, a known sequence may be considered to be a match if a candidatesequence and the known sequence are at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, or even100% identical. Typically the percent identity will be evaluated over awindow of at least 10 nucleotides in length, e.g, 10-15 nucleotides,15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, etc. The lengthof the window may be selected according to a variety of differentcriteria including, but not limited to, the number of sequences in theplurality of known sequences, the identity or source of the plurality ofknown sequences, etc. For example, if a candidate sequence is beingcompared against sequences in a large database such as GenBank, it maybe desirable to use a longer length than if a database containing fewersequences is used. In certain embodiments of the invention sequences arecompared across a plurality of different windows, not necessarilyadjacent to one another. Preferably the combined length of the windowsis at least 10 nucleotides in length, e.g, 10-15 nucleotides, 15-20nucleotides, 20-25 nucleotides, 25-30 nucleotides, etc. In someinstances multiple sequences in the set of known sequences may match.The sequences may, for example, represent homologous genes found in thesame organism as that from which the template was derived, homologousgenes from different organisms, pseudogenes, cDNA and genomic sequences,etc.

In general, the candidate sequence that most closely resembles asequence in the set of known sequences is selected as correct.Alternately, e.g., if there is reason to believe that the sequencingmethod may have been subject to a high error rate it may be preferableto select the corresponding sequence from the database as correct. Forexample, if the error rate is known to be above a predeterminedthreshold it may be preferable to select a sequence from the database asthe correct sequence.

The length required in order to ensure that the likelihood of matchesbeing found for multiple candidate sequences will depend on a variety ofconsiderations including, but not limited to, the particular set ofknown sequences, the threshold for accepting matches, etc. In general, asequence of length ˜25-26 nucleotides would only be represented once inthe genome of a typical organism. Therefore generating candidatesequences of approximately this length is sufficient to identify thecorrect sequence. In general, the candidate sequence should be at least10 nucleotides in length, preferably at least 15, at least 20nucleotides in length, e.g., between 20-25, 25-30, 30-35, 35-40, 45-50,or even longer.

Sequence Identification by Comparing a First Set of Candidate Sequenceswith a Second Set of Candidate Sequences

In certain embodiments of the invention decoding is performed bygenerating a first ordered list of probe families using a firstcollection of probe families encoded according to a first encoding,generating a first set of candidate sequences therefrom and thengenerating a second ordered list of probe families from the sametemplate using a second collection of probe families encoded accordingto a second encoding and generating a second set of candidate sequencestherefrom. The newly synthesized DNA strand is removed from the templatebetween the two sequencing reactions, or a template of identicalsequence is sequenced using the second collection of probe families. Thesets of candidate sequences are compared. It will be appreciated thatregardless of which collection of probe families is used, one of thecandidate sequences will be the correct sequence while the others arenot correct (or are at best partially correct). Thus every set ofcandidate sequences will contain the correct sequence, but in most casesthe other candidate sequences in any given set candidate sequences willdiffer from those found in another set of candidate sequences.Therefore, by simply comparing the two sets of candidate sequences, thecorrect sequence can be determined. It is not necessary to generatecandidate sequences of equal length using the two differently encodedcollections of probe families. In preferred embodiments of the inventionthe candidate sequences generated using the second collection of probefamilies can be as short as 2 nucleotides or, equivalently, the orderedlist of probe families generated using the second collection of probefamilies can be as short as 1 element (i.e., a single cycle of ligationand detection).

FIGS. 31A-31C show an example of candidate sequence generation anddecoding using two distinguishably labeled preferred probe families.FIG. 31A shows a preferred collection of probe families encodedaccording to a first encoding. FIG. 31B shows generation of 4 candidatesequences from the ordered list of probe families Yellow, Green, Red,Blue (which could be represented as “2314” in which Red=1, Yellow=2,Green=3, and Blue=4), of which the correct sequence is assumed to beCAGGC (shown in bold). FIG. 31C shows a preferred collection of probefamilies encoded according to a second encoding. Since the firstdinucleotide in the template is CA, the uppermost probe in the Yellowprobe family will ligate to the extendable terminus in the first cycleof extension. This results in the following set of candidate sequencesfor the first dinucleotide: CA, TC, GG, AT. Among the candidatesequences generated using the first collection of probe families, onlythe sequence CAGGC begins with any of these dinucleotides. Therefore itmust be the correct sequence. In general, it is preferred that the firstand second collections of probe families should fulfill the followingcriteria: When the first and second collections of probe families arecompared, (i) 3 of the 4 probes in each of the probe families in thefirst collection should be assigned to a new probe family in the secondcollection; and (ii) each of the 3 reassigned probes should be assignedto a different probe family in the second collection.

Using a Known Nucleotide Identity to Decode an Ordered List of ProbeFamilies

As described above, candidate sequences can be generated by assuming anidentity for a single nucleotide in the extended duplex or template.Depending on the specific probe family collection used, it willgenerally be necessary to generate at least 4 candidate sequences.However, generation of multiple candidate sequences can be avoided ifthe identity of at least one nucleotide in the template (and thereforealso in the extended duplex) is known. In that case, it will only benecessary to generate a single candidate sequence. The method forgenerating the candidate sequence is identical to that described above.The identity of the at least one nucleotide in the template may bedetermined using any sequencing method including, but not limited tosequencing methods A, primer extension from an initializingoligonucleotide using a set of distinguishably labeled nucleotides and apolymerase, etc. It will be appreciated that one or more nucleotides inthe template can first be sequenced using a sequencing method other thansequencing method AB, and the initializing oligonucleotide and anyextension products can then be removed, and the same template subjectedto sequencing using sequencing methods AB (or vice versa).

Another approach is to simply sequence a template that contains one ormore known nucleotides of known identity in addition to a portion whosesequence is to be determined. For example, the portion of the templatebetween the region to which the initializing oligonucleotide binds andat which the unknown sequence begins can include one or more nucleotidesof known identity. By subjecting this portion of the template tosequencing methods AB, the identity of one or more nucleotides in thesequence will be predetermined and can thus be used to generate a singlecandidate sequence, which will be the correct sequence.

The methods described above therefore comprise steps of (i) assigning anidentity to a nucleotide in the template adjacent to a nucleotide ofknown identity by determining which identity is consistent with theidentity of the known nucleotide and the possible sequences of theconstrained portion of the probe whose proximal nucleotide ligatedopposite the nucleotide adjacent to the nucleotide of known identity;(ii) assigning an identity to a succeeding nucleotide by determiningwhich identity is consistent with possible sequences of the constrainedportion of the probe whose proximal nucleotide ligated opposite thesucceeding nucleotide; and (iii) repeating step (ii) until the sequenceis determined. It is to be understood that these steps are equivalent toperforming the same steps on the extended duplex since there is aprecise correspondence between the extended duplex and the region of thetemplate to be sequenced.

Sequencing with Less Preferred Probe Families

Less preferred collections of probe families may be used to performsequencing methods AB in a similar manner to the way in which preferredcollections of probe families are used. However, the results may differin a number of respects. For example, certain portions of the sequencemay be fully identified from the candidate sequences without the needfor additional information. FIG. 32 shows an example of sequencedetermination using a less preferred collection of probe familiesencoded as shown in FIG. 28. Sequence determination generally proceedsas described for preferred collections of probe families. The templateof interest has the sequence “GCATGA”, which results in “12341” as theordered list of probe families. Assuming that the nucleotide at position1 is A yields “ACATGA” as a candidate sequence. However, unlike the casewith the preferred collections of probe families, there are twopossibilities for the second nucleotide since the label “1” isassociated with two different dinucleotides that have A as the firstnucleotide, i.e., “AA” and “AG”. Thus assuming that the nucleotide atposition 1 is A yields “ACATGC” as a second candidate sequence. Assumingthat the nucleotide at position 1 is G yields “GCATGA” as a candidatesequence and also yields “GCATGC” as a candidate sequence. Since thelabel “1” is not associated with any dinucleotides that have C or T atposition 1, no candidate sequences beginning with “C” or “T” aregenerated. FIG. 32 shows the 4 candidate sequences aligned with eachother. It will be observed that the middle 4 nucleotides of all thecandidate sequences are CATG. Therefore, the correct sequence mustinclude CATG at positions 2-5. If only these nucleotides are ofinterest, there is no need to perform further decoding steps.

As mentioned above, collections of probe families need not consist offour different probe families but can consist of any number greater than2, up to 4^(N), where N is the length of the constrained portion.However, if fewer than 4 families are used it may be necessary togenerate more than 4 candidate sequences, while if more than 4 probefamilies are used additional labels will be required. For these andother reasons collections consisting of 4 probe families are preferred.

Sequence Identification by Comparing Candidate Sequences with Each Other

In certain embodiments of the invention part or all of a sequence ofinterest may be determined by comparing candidate sequences with eachother. In general, such a comparison may not be sufficient to determinewhich of the candidate sequences is correct across its entire length.However, if two or more of the candidate sequences are identical orsufficiently similar over a portion of the sequences, this informationmay be sufficient to explicitly identify the sequence of nucleotides inthe template within that portion as described above.

If desired, the template can be sequenced one or more additional timesusing alternatively encoded probe families to yield additional portionswith an identified sequence. These portions can be combined to assemblea sequence of a desired length.

Error Correction Using Probe Families. It is often desirable to sequencemultiple templates that represent all or part of the same DNA sequenceand to align the sequences. If the templates contain only part of aregion of interest, a longer sequence is then obtained by assemblingoverlapping fragments. For example, when sequencing the genome of anorganism, typically the DNA is fragmented, and enough fragments aresequenced so that each stretch of DNA is represented in several (e.g.,4-12) different fragments. Computer software for assembling overlappingsequences into a longer sequence is known to one of skill in the art.

When conventional sequencing methods are used, it is frequently the casethat multiple fragments align perfectly over a region except that one ofthe fragments (referred to as an anomalous fragment) differs from theothers at a single position within the region. Determining whether theisolated difference represents a sequencing error or whether a genuinedifference (e.g., a single nucleotide polymorphism) exists at theposition the can be problematic.

The invention provides novel methods of performing error checking usingsequencing methods AB. According to the method, templates comprisingfragments that represent the same stretch of DNA are sequenced using acollection of distinguishably labeled probe families as described above,resulting in an ordered list of probe families for each template. Theordered lists of probe families are aligned. If several lists alignperfectly over a predetermined length, e.g, 10, 15, 20, or 25 or moreelements in the lists, except for one list that differs at a singleposition from the other fragments, the difference is ascribed to asequencing error. If an actual polymorphism exists, the ordered probelist generated from the anomalous fragment will differ at two or moreadjacent positions from the ordered probe lists generated from the otherfragments.

For example, applying sequencing methods AB using a preferred collectingof probe families that uses encoding 4 in Table 1 to a templatecomprising the sequence 5′-CAGACGACAAGTATAATG-3′ (SEQ ID NO. 1) yieldsthe following ordered list of probe families: “23324322132444142”, asshown below:

23324322132444142 (SEQ ID NO. 1) CAGACGACAAGTATAATG

If there is an actual SNP (e.g., CAGACGAGAAGTATAATG (SEQ ID NO. 2)), inwhich the underlined nucleotide represents the polymorphic site), itresults in changes in two consecutive elements in the list:23324333132444142, in which underlining indicates the change that occursas a result of the SNP. The correspondence between the ordered list ofprobe families and sequence containing a SNP is shown below:

23324333132444142 (SEQ ID NO. 2) CAGACGAGAAGTATAATG

However, an error in identifying the label associated with a ligatedextension probe results in a single error in the ordered list of probefamilies and a change in the resulting candidate sequence from thatpoint forward. For example, an error in determining the label associatedwith the 7^(th) ligated extension probe 23324332132444142 (in which theunderlined number represents the misidentified label) changes theresulting candidate sequence to CAGACGAGTTCATATTAC (SEQ ID NO. 3), inwhich the underlined portion indicates the change that occurs as aresult of the sequencing error. The correspondence between the orderedlist of probe families and the sequence is shown below:

23324332132444142 (SEQ ID NO. 3) CAGACGAGTTCATATTAC

When using a 3 base, 4 label scheme, a fragment that contains a SNPresults in 3 consecutive differences in the ordered list of probefamilies for the anomalous fragment, while a sequencing error results inonly 1 difference. For example, when the collection of probe familiesencoded as shown in FIG. 29 is used, an ordered list of probe familyidentities for the sequence CAGACGACAAGTATAATG (SEQ ID NO. 1) is shownbelow:

2322224132412244 (SEQ ID NO. 1) CAGACGACAAGTATAATG

An anomalous fragment containing a SNP, e.g., CAGACGAGAAGTATAATG (SEQ IDNO. 2), would result in an ordered list of probe families that differsat 3 consecutive positions relative to ordered lists generated fromfragments that do not contain the SNP, as shown below:

2322213332412244 (SEQ ID NO. 2) CAGACGAGAAGTATAATG

A sequencing error would result in only a single difference in theordered list of probe families and would result in a completelydifferent generated candidate sequence from the point of the errorforward.

Thus when an ordered list of probe families generated from a fragment(an anomalous fragment) aligns with ordered lists of probe familiesgenerated from other fragments that represent the same stretch of DNAbut differs from the other ordered lists at a single isolated position,it is likely that the ordered list containing the difference representsa sequencing error (misidentification of a probe family). When anordered list of probe families generated from a fragment (an anomalousfragment) aligns with ordered lists of probe families generated fromother fragments that represent the same stretch of DNA but differs fromthe other ordered lists at 2 or more consecutive positions, it is likelythat the anomalous fragment contains a SNP. Preferably the alignedportions of the ordered lists of probe families are at least 3 or 4elements in length, preferably at least 6, 8, or more elements inlength. Preferably the aligned portions are at least 66% identical, atleast 70% identical, at least 80% identical, at least 90% identical, ormore, e.g., 100% identical.

Similarly, when a candidate sequence for a fragment aligns withcandidate sequences for other fragments that represent the same stretchof DNA over a first portion of the sequence but differs substantiallyfrom candidate sequences for other fragments over a second portion ofthe sequence, is it likely that a sequencing error occurred. When acandidate sequence for a fragment aligns with candidate sequences forother fragments that represent the same stretch of DNA over two portionsof the sequence but differ at a single position, it is likely that theanomalous fragment contains a SNP. Preferably the aligned portions ofthe candidate sequences are at least 4 nucleotides in length. Preferablythe aligned portions are at least 66% identical, at least 70% identical,at least 80% identical, at least 90% identical, or more, e.g., 100%identical.

The invention therefore provides a method of distinguishing a singlenucleotide polymorphism from a sequencing error comprising steps of: (a)sequencing a plurality of templates using sequencing methods AB, whereinthe templates represent overlapping fragments of a single nucleic acidsequence; (b) aligning the sequences obtained in step (a); and (c)determining that a difference between the sequences represents asequencing error if the sequences are substantially identical across afirst portion and substantially different across a second portion, eachportion having a length of at least 3 nucleotides. The invention furtherprovides a method of distinguishing a single nucleotide polymorphismfrom a sequencing error comprising steps of: (a) obtaining a pluralityof ordered lists of probe families by performing sequencing methods ABusing a plurality of templates that represent overlapping fragments of asingle nucleic acid sequence; (b) aligning the ordered lists of probefamilies obtained in step (a) to obtain an aligned region within whichthe lists are at least 90% identical; and (c) determining that adifference between the ordered lists of probe families represents asequencing error if the lists differ at only one position within thealigned region; or (d) determining that a difference between the orderedlists of probe families represents a single nucleotide polymorphism ifthe lists differ at two or more consecutive positions within the alignedregion.

Delocalized Information Collection

As is well known in the art, a “bit” (binary digit) refers to a singledigit number in base 2, in other words, either a 1 or a zero, andrepresent the smallest unit of digital data. Since a nucleotide can haveany of 4 different identities, it will be appreciated that specifyingthe identity of a nucleotide requires 2 bits. For example, A, G, C, andT could be represented as 00, 01, 10, and 11, respectively. Specifyingthe name of a probe family in a preferred collection of distinguishablylabeled probe families requires 2 bits since there are fourdistinguishably labeled probe families.

In most conventional forms of sequencing, and in sequencing methods A,each nucleotide is identified as a discrete unit, and informationcorresponding to one nucleotide at a time is gathered. Each detectionstep acquires two bits of information from a single nucleotide. Incontrast, sequencing methods AB acquire less than two bits ofinformation from each of a plurality of nucleotides in each detectionstep while still acquiring 2 bits of information per detection step whena preferred collection of probe families is used. Each probe family namein an ordered list of probe families represents the identity of at least2 nucleotides in the template, with the exact number being determined bythe length of the sequence determining portion of the probes. Forexample, consider the ordered list of probe families obtained from thesequence 5′-CAGACGACAAGTATAATG-3′ (SEQ ID NO. 1) using a collection ofprobe families encoded according to encoding 4 in Table 1:

23324322132444142 (SEQ ID NO. 1) CAGACGACAAGTATAATG 

Probe family 2 is the first probe family in the list since thedinucleotide CA is one of the specified portions present in probes ofprobe family 2. Probe family 3 is the second probe family in the listsince the dinucleotide AG is one of the specified portions present inprobes of probe family 3. As mentioned above, since there are 4 probefamilies, each probe family identity represents 2 bits of information.Thus each detection step gathers 2 bits of information about 2nucleotides, resulting in an average of 1 bit of information from eachnucleotide.

The invention therefore provides a method for determining a sequence,wherein the method comprises multiple cycles of extension, ligation, anddetection, and wherein the detecting step comprises simultaneouslyacquiring an average of two bits of information from each of at leasttwo nucleotides in the template without acquiring two bits ofinformation from any individual nucleotide. The invention furtherprovides a method for determining a sequence of nucleotides in atemplate polynucleotide using a first collection of oligonucleotideprobe families, the method comprising the steps of: (a) performingsequential cycles of extension, ligation, detection, and cleavage,wherein an average of two bits of information are simultaneouslyacquired from each of at least two nucleotides in the template duringeach cycle without acquiring two bits of information from any individualnucleotide; and (b) combining the information obtained in step (a) withat least one bit of additional information to determine the sequence. Invarious embodiments of the invention the at least one bit of additionalinformation comprises an item selected from the group consisting of: theidentity of a nucleotide in the template, information obtained bycomparing a candidate sequence with at least one known sequence; andinformation obtained by repeating the method using a second collectionof oligonucleotide probe families.

Thus while the methods do not acquire 2 bits of information fromindividual nucleotides, an average of 2 bits of information is gatheredfrom the template in each cycle, but in a delocalized manner whenpreferred collections of probe families are used. When using collectionsof 2 or 3 probe families, less than 2 bits of information are gatheredduring each cycle.

Delocalized information collection has a number of advantages includingallowing the application of error checking methods such as thosedescribed above. In addition, since each nucleotide in the template isinterrogated more than once in preferred embodiments, delocalizedinformation collection can help avoid systematic biases in detectingfluorophores associated with particular nucleotides.

The probe families and collections of probe families described hereincan be used in a variety of sequencing methods in addition to methodsthat involve successive cycles of extension, ligation, and cleavage ofthe probe. The invention also provides probe families and collections ofprobe families having the sequences and structures as described above,wherein the probes optionally do not contain a scissile linkage. Forexample, the probes can contain only phosphodiester backbone linkagesand/or may not contain a trigger residue. In some embodiments of theinvention the probe families are used to perform sequencing usingsuccessive cycles of extension and ligation, but not involving cleavageduring each cycle. For example, the probe families can be used in aligation-based method such as that described in WO2005021786 andelsewhere in the art. To use the probe families in such a method, thelabel on the probe should be attached by a cleavable linker, e.g., asdisclosed in WO2005021786, such that it can be removed without cleavinga scissile linkage of the nucleic acid. Such a method can be used togenerate an ordered list of probe families, e.g., by performing multiplereactions in parallel or sequentially, using the probe families ratherthan the ligation cassettes described in WO2005021786, and thenassembling the list of probe families. The list is decoded as describedabove.

I. Kits

A variety of kits may be provided for carrying out different embodimentsof the invention. Certain of the kits include extension oligonucleotideprobes comprising a phosphorothiolate linkage. The kits may furtherinclude one or more initializing oligonucleotides. The kits may containa cleavage reagent suitable for cleaving phosphororothiolate linkages,e.g., AgNO₃ and appropriate buffers in which to perform the cleavage.Certain of the kits include extension oligonucleotide probes comprisinga trigger residue such as a nucleoside containing a damaged base or anabasic residue. The kits may further include one or more initializingoligonucleotides. The kits may contain a cleavage reagent suitable forcleaving a linkage between a nucleoside and an adjacent abasic residueand/or a reagent suitable for removing a damaged base from apolynucleotide, e.g., a DNA glycosylase. Certain kits containoligonucleotide probes that comprise a disaccharide nucleotide andcontain periodate as a cleavage reagent. In certain embodiments the kitscontain a collection of distinguishably labeled oligonucleotide probefamilies.

Kits may further include ligation reagents (e.g., ligase, buffers, etc.)and instructions for practicing the particular embodiment of theinvention. Appropriate buffers for the other enzymes that may be used,e.g., phosphatase, polymerases, may be included. In some cases, thesebuffers may be identical. Kits may also include a support, e.g. magneticbeads, for anchoring templates. The beads may be functionalized with aprimer for performing PCR amplification. Other optional componentsinclude washing solutions; vectors for inserting templates for PCRamplification; PCR reagents such as amplification primers, thermostablepolymerase, nucleotides; reagents for preparing an emulsion; reagentsfor preparing a gel, etc.

In certain preferred kits, fluorescently labeled oligonucleotide probescomprising phosphorothiolate linkages are provided such that probescorresponding to different terminal nucleotides of the probe carrydistinct spectrally resolvable fluorescent dyes. More preferably, foursuch probes are provided that allow a one-to-one correspondence betweeneach of four spectrally resolvable fluorescent dyes and the fourpossible terminal nucleotides of a probe.

An identifier, e.g., a bar code, radio frequency ID tag, etc., may bepresent in or on the kit. The identifier can be used, e.g., to uniquelyidentify the kit for purposes of quality control, inventory control,tracking, movement between workstations, etc.

Kits will generally include one or more vessels or containers so thatcertain of the individual reagents may be separately housed. The kitsmay also include a means for enclosing the individual containers inrelatively close confinement for commercial sale, e.g., a plastic box,in which instructions, packaging materials such as styrofoam, etc., maybe enclosed.

J. Automated Sequencing Systems

The invention provides a variety of automated sequencing systems thatcan be used to gather sequence information from a plurality of templatesin parallel, i.e., substantially simultaneously. Preferably thetemplates are arrayed on a substantially planar substrate. FIG. 21 showsa photograph of one of the inventive systems. As shown in the upper partof the photograph, the inventive system comprises a CCD camera, afluorescence microscope, a movable stage, a Peltier flow cell, atemperature controller, a fluid handling device, and a dedicatedcomputer. It will be appreciated that various substitutions of thesecomponents can be made. For example, alternative image capture devicescan be used. Further details of this system are provided in Example 9.

It will be appreciated that the inventive automated sequencing systemand associated image processing methods and software can be used topractice a variety of sequencing methods including both theligation-based methods described herein and other methods including, butnot limited to, sequencing by synthesis methods such as fluorescence insitu sequencing by synthesis (FISSEQ) (see, e.g., Mitra R D, et al.,Anal Biochem., 320(1):55-65, 2003). As is the case for theligation-based sequencing methods described herein, FISSEQ may bepracticed on templates immobilized directly in or on a semi-solidsupport, templates immobilized on microparticles in or on a semi-solidsupport, templates attached directly to a substrate, etc.

One important aspect of the inventive system is a flow cell. In general,a flow cell comprises a chamber that has input and output ports throughwhich fluid can flow. See, e.g., U.S. Pat. Nos. 6,406,848 and 6,654,505and PCT Pub. No. WO98053300 for discussion of various flow cells andmaterials and methods for their manufacture. The flow of fluid allowsvarious reagents to be added and removed from entities (e.g., templates,microparticles, analytes, etc.) located in the flow cell.

Preferably a suitable flow cell for use in the inventive sequencingsystem comprises a location at which a substrate, e.g. a substantiallyplanar substrate such as a slide, can be mounted so that fluid flowsover the surface of the substrate, and a window to allow illumination,excitation, signal acquisition, etc. In accordance with the inventivemethods, entities such as microparticles are typically arrayed on thesubstrate before it is placed within the flow cell.

In certain embodiments of the invention the flow cell is verticallyoriented, which allows air bubbles to escape from the top of the flowcell. The flow cell is arranged such that the fluid path runs frombottom to top of the flow cell, e.g., the input port is at the bottom ofthe cell and the output port is at the top of the cell. Since anybubbles that may be introduced are buoyant, they rapidly float to theoutput port without obscuring the illumination window. This approach, inwhich gas bubbles are allowed to rise to the surface of a liquid byvirtue of their lower density relative to that of the liquid is referredto herein as “gravimetric bubble displacement”. Thus the inventionprovides a sequencing system comprising a flow cell oriented so as toallow gravimetric bubble displacement. Preferably the substrate havingmicroparticles directly or indirectly attached thereto (e.g., covalentlyor noncovalently linked to the substrate) or immobilized in or on asemi-solid support that is adherent to or affixed to the substrate ismounted vertically within the flow cell, i.e., the largest planarsurface of the substrate is perpendicular to the ground plane. Since inpreferred embodiments the microparticles are immobilized in or on asupport or substrate, they remain at substantially fixed positions withrespect to one another, which facilitates serial acquisition of imagesand image registration.

FIGS. 24A-J shows schematic diagrams of inventive flow cells or portionsthereof, in various orientations. The inventive flow cells can be usedfor any of a variety of purposes including, but not limited to, analysismethods (e.g., nucleic acid analysis methods such as sequencing,hybridization assays, etc.; protein analysis methods, binding assays,screening assays, etc. The flow cells may also be used to performsynthesis, e.g., to generate combinatorial libraries, etc.

FIG. 22 shows a schematic diagram of another inventive automatedsequencing system. The flow cell is mounted on a temperature-controlled,automated stage (similar to the one described in Example 9) and isattached to a fluid handling system, such as a syringe pump with amulti-port valve. The stage accommodate multiple flow cells in order toallow one flow cell to be imaged while other steps such as extension,ligation, and cleavage are being performed on another flow cell. Thisapproach maximizes utilization of the expensive optical system whileincreasing the throughput.

The fluid lines are equipped with optical and/or conductance sensors todetect bubbles and to monitor reagent usage. Temperature control andsensors in the fluidics system assure that reagents are maintained at anappropriate temperature for long term stability but are raised to theworking temperature as they enter the flow cell to avoid temperaturefluctuations during the annealing, ligation and cleavage steps. Reagentsare preferably pre-packaged in kits to prevent errors in loading.

The optics includes four cameras—each taking one image through one offour filter sets. In order to reduce the effects of photobleaching, theillumination optics may be engineered to illuminate only the area beingimaged, to avoid multiple illumination of the edges of the fields. Theimaging optics may be built from standard infinity-corrected microscopeobjectives and standard beam-splitters and filters. Standard 2,000×2,000pixel CCD cameras can be used to acquire the images. The systemincorporates appropriate mechanical supports for the optics.Illumination intensity is preferably monitored and recorded for lateruse by the analysis software.

In order to rapidly acquire a plurality of images (e.g., approximately1800 or more non-overlapping image fields in a representativeembodiment), the system preferably uses a fast autofocus system.Autofocus systems based on analysis of the images themselves are wellknown in the art. These generally require at least 5 frames per focusingevent. This is both slow and costly in terms of the extra illuminationrequired to acquire the focusing images (increases photobleaching). Incertain embodiments of the invention an alternate autofocusing system isused, e.g., a system based on independent optics that can focus asquickly as the mechanical systems can respond. Such systems are known inthe art and include, for examples the focusing systems used in consumerCD players, which maintain sub-micron focusing in real time as the CDspins.

In certain embodiments of the invention the system is operated remotely.Scripts for implementing specific protocols may be stored in a centraldatabase and downloaded for each sequencing run. Samples can be barcodedto maintain integrity of sample tracking and associating samples withthe final data. Central, real-time monitoring will allow quickresolution of process errors. In certain embodiments images gathered bythe instruments will immediately be uploaded to a central,multi-terabyte storage system and a bank of one or more processor(s).Using tracking data from the central database, the processor(s) analyzethe images and generate sequence data and, optionally, process metrics,such as background fluorescence levels and bead density, in order, e.g.,to track instrument performance.

Control software is used to properly sequence the pumps, stage, cameras,filters, temperature control and to annotate and store the image data. Auser interface is provided, e.g., to assist the operator in setting upand maintaining the instrument, and preferably includes functions toposition the stage for loading/unloading slides and priming the fluidlines. Display functions may be included, e.g., to show the operatorvarious running parameters, such as temperatures, stage position,current optical filter configuration, the state of a running protocol,etc. Preferably an interface to the database to record tracking datasuch as reagent lots and sample IDs is included.

K. Image and Data Processing Methods

The invention provides a variety of image and data processing methodsthat may be implemented at least in part as computer code (i.e.,software) stored on a computer readable medium. Further details arepresented in Examples 9 and 10. In addition, in general, both sequencingmethods A and B generally employ appropriate computer software toperform the processing steps involved, e.g., keeping track of datagathered in multiple sequencing reactions, assembling such data,generating candidate sequences, performing sequence comparisons, etc.

L. Computer-readable Media Storing Sequence Information

In addition, the invention provides a computer-readable medium thatstores information generated by applying the inventive sequencingmethods. Information includes raw data (i.e., data that has not beenfurther processed or analyzed), processed or analyzed data, etc. Dataincludes images, numbers, etc. The information may be stored in adatabase, i.e., a collection of information (e.g., data) typicallyarranged for ease of retrieval, for example, stored in a computermemory. Information includes, e.g., sequences and any informationrelated to the sequences, e.g., portions of the sequence, comparisons ofthe sequence with a reference sequence, results of sequence analysis,genomic information, such as polymorphism information (e.g., whether aparticular template contains a polymorphism) or mutation information,etc., linkage information (i.e., information pertaining to the physicallocation of a nucleic acid sequence with respect to another nucleic acidsequence, e.g., in a chromosome), disease association information (i.e.,information correlating the presence of or susceptibility to a diseaseto a physical trait of a subject, e.g., an allele of a subject), etc.The information may be associated with a sample ID, subject ID, etc.Additional information related to the sample, subject, etc., may beincluded, including, but not limited to, the source of the sample,processing steps performed on the sample, interpretations of theinformation, characteristics of the sample or subject, etc. Theinvention also includes a method comprising receiving any of theaforesaid information in a computer-readable format, e.g., stored on acomputer-readable medium. The method may further include a step ofproviding diagnostic, prognostic, or predictive information based on theinformation, or a step of simply providing the information to a thirdparty, preferably stored on a computer-readable medium.

The following examples are provided for illustrative purposes and arenot intended to limit the invention.

EXAMPLE 1 Efficient Cleavage and Ligation of PhosphorothiolatedOligonucleotides

This example describes an experiment demonstrating efficient ligationand cleavage of extension oligonucleotides containing a 3′-Sphosphorothiolate linkage.

Materials and Methods

Ligation Sequencing Protocol

Template Preparation: To demonstrate evaluate the potential ofsequencing by cycled oligonucleotide ligation and cleavage and toexplore the effect of variations in certain aspects of the method, twosets of model bead-based template populations were prepared. Inpreferred implementations, as described in the Examples, cycledoligonucleotide ligation and cleavage extends strands in the 3′→5′direction. Therefore, to evaluate ligation efficiencies, model templateswere bound to beads at the 5′ end and designed with the same bindingregion at the 3′ end. One set was comprised of short (70 bp)oligonucleotides bound to streptavidin-coated magnetic beads (1 micron)via a dual biotin moiety. Each of these short template populations weredesigned with an identical primer binding region (40 bp) and a uniquesequence region (30 bp) at the 3′ end. The short oligonucleotidetemplate populations were termed ligation sequencing templates 1-7(LST1-7).

The second set of bead-based template populations were designed fromlong, PCR-generated DNA fragments (232-bp) derived by inserting 183-bpof spacer sequence (from a human p53 exon) into each templatepopulation. Templates were amplified with dual biotin-containing forwardprimers and reverse primers containing the same 30 base unique 3′ endsequence as the short template populations. The templates were madesingle-stranded by melting off one of the strands with sodiumhydroxide-containing buffer. These long template populations weredesigned to mimic the species generated from short-fragment paired-endlibraries described in a copending patent application and were termedlong-LST1-7.

Primer Hybridization: 2.5 μL of 100 μM FAM-labeled primer was premixedwith 100 μL 1× Klenow Buffer. This solution was added to a 30 μL aliquotof magnetic beads (10⁶/μL) with attached template after removal of thebuffer, and the resulting solution was well mixed. After allowingtemplate/primer hybridization to occur (hybridization reaction wascarried out for 2 minutes at 65° C., 2 minutes at 40° C. and 2 minuteson ice), the primer/buffer was removed, and the beads were washed using3× Wash 1E buffer, and then resuspended in 300 μL (10⁶/mL) in TENTbuffer (containing 10 mM Tris, 2 mM EDTA, 30 mM NaOAc, and 0.01% TritonX-100).

Ligation 1: 2.5×10⁶ LST7 beads with hybridized LigSeq-FAM were thenincubated for 30 minutes at 37° C. in a mixture containing 1 μL, of 100μM LST7-1 Nonamer, 4 μL 5× T4 Ligase Buffer (Invitrogen), 14 μL, of H₂Oand 1 μL, of T4 Ligase (1 u/μL, Invitrogen).

Cleavage 1: The beads were then washed 3 times with 100 μL of LSWash1(containing 1×TE, 30 mM sodium acetate, 0.01% Triton X100); a 10μL-aliquot of this solution was removed and saved for analysis. Thebeads (1×) were then washed in 100 μL of 30 mM sodium acetate. 50 μL of50 mM AgNO₃ was added to this solution and the resulting mixture wasincubated at 37° C. for 20 minutes. AgNO₃ was removed, and the beadswere washed once in 100 μL of 30 mM sodium acetate. The beads were thenwashed in 3 times with 100 μL of LSWash1, resuspended in 90 μL Wash(TENT buffer); and a 10 μL-aliquot of this solution was removed andsaved for analysis.

Ligation 2: After removal of the TENT buffer, the beads were resuspendedin 14 μL of H₂O, and incubated at 37° C. for 30 minutes with a mixturecontaining 1 μL, of 100 μM LST7-5 Nonamer, 4 μL of 5× T4 Ligase Buffer(Invitrogen) and 1 μL of T4 Ligase (1 u/μL, Invitrogen).

Cleavage 2: The beads were washed 3 times in 100 μL of LSWash1 (1×TE, 30mM sodium acetate, 0.01% Triton X100), and resuspended in 45 μL Wash1E.A 15 μL-aliquot of this mixture was removed and saved for analysis. Thebeads were then washed once with 100 μL of 30 mM sodium acetate andresuspended in 5 μL of 20 mM sodium acetate. 50 μL of 50 mM AgNO₃ wasadded to the beads and the mixture was incubated at 37° C. for 20minutes. After removal of AgNO₃, the beads were washed once with 100 μLof 30 mM sodium acetate. The beads were then washed three times in 100μL of LSWash1, and resuspended in 30 μL Wash1E. A 20 μL-aliquot of thismixture was removed and saved for analysis.

Results

The experiment will be better understood with reference to FIG. 8. Theupper section of FIG. 8 shows an overall outline of the experimentalprocedure. An initializing oligonucleotide (primer) was hybridized to atemplate (designated LST7), which was attached to a bead via a biotinlinkage. The initializing oligonucleotide contained a 5′ phosphate andwas fluorescently labeled with FAM at its 3′ end. Two 9mer (nonamer)oligonucleotide probes (1^(st) cleavable oligo and 2^(nd) cleavableoligo) were synthesized to contain an internal phosphorothiolatedthymidine base (sT) (underlined). The first cleavable probe was ligatedto the extendable terminus of the primer using T4 DNA ligase and wasthen cleaved using silver nitrate. Cleavage removed the terminal 5nucleotides of the extension probe and generated an extendable terminuson the portion of the probe that remained ligated to the primer. Thesecond cleavable probe was then ligated to the extendable terminus andwas then similarly cleaved.

A fluorescent capillary electrophoresis gel shift assay was used tomonitor steps of ligation and cleavage. In this assay, the primer ishybridized to a template strand such that the 5′ phosphate can serve asa ligation substrate for incoming oligonucleotide probes (thefluorophore serves as a reporter for mobility-based capillary gelelectrophoresis). After each step an aliquot of beads was removed foranalysis. Following ligation of oligonucleotide probes, the magneticbeads were collected using a magnet and the ligated species consistingof the primer and probe(s) ligated thereto was released from thetemplate beads by heat denaturation and subjected to fluorescentcapillary electrophoresis using an automated DNA sequencing instrument(ABI 3730) with labeled size standards (lissamine ladder; size range15-120 nucleotides; appears as a set of orange peaks in chromatograms,see FIG. 8). In a typical gel shift, the potential peaks include, i)primer peaks (due to no extension or the lack of primer extension), ii)adenylation peaks (due to the attachment of an adenosine residue at the5′ end of a nonproductive ligation junction by the action of DNAligase—see mechanism in FIG. 8F, see also Lehman, I. R., Science,186:790-797, 1974), and iii) completion peaks (due to the attachment ofan oligo probe). One benefit of using gel shift assays to evaluateligation efficiency is that the areas under the peaks directly correlatewith the concentration of each species.

FIG. 8A shows a control ligation performed using T4 DNA ligase and anexact match probe containing only phosphodiester linkages (shown to theleft of FIG. 8A). Orange peaks represent size markers. The blue peak atthe left indicates the position of the primer in the absence ofligation. Ligation of the exact match probe results in a shift to theleft (arrow). FIG. 8B shows a ligation performed under the sameconditions using a probe containing an internal thiolated T base (shownto the left of FIG. 8B). A shift identical to that observed with thecontrol probe was seen (arrow). Bead-linked template populationscontaining the ligated phosphorothiolated probes were then incubatedwith silver nitrate to induce probe cleavage. Gel-shift analysisconfirmed efficient cleavage by demonstration of a left-shifted, 4-bpcleavage product (FIG. 8C). The expected cleavage product is shown tothe left of FIG. 8C. Cleaved bead-based template populations were thenexposed to a second round of ligation and demonstrated productiveligation by the appearance of a right-shifted, 13-bp extension product(FIG. 8D). The expected cleavage product is shown to the left of FIG.8D. A second round of cleavage confirmed efficient multiple cleavagesteps could be accomplished as demonstrated by the expectedleft-shifted, 8-bp cleavage product (FIG. 8E).

These results demonstrate successful ligation and cleavage of probescontaining phosphorothiolate linkages.

It is evident that ligation did not proceed to 100% completion in theseexperiments, although a greater degree of completion was observed inother experiments using T4 DNA ligase (see below). While it is certainlydesirable that the ligation proceed to completion it is not arequirement. For example, it is possible to effectively “cap” anyunligated 5′ ends by treating with a 5′-phosphatase after the ligationstep as described above. In that case, however, there would be a limitto the number of sequential ligations that could be performed, due toattrition of ligatable molecules. With a given number of sequentialligations, the read length will depend on the length of the proberemaining after each ligation/cleavage cycle and on the number ofsequencing reactions, each followed by removal of the primer andhybridization of a primer that binds to a different portion of theprimer binding site, that can be performed on a given template, alsoreferred to as the number of “resets”). This argues for the use oflonger probes with the cleavable linkage located towards the 5′ end ofthe probe. In our experiments, hexamer probes lead to greater amounts ofun-ligatable adenylation products than octamers and longer probes. Thusoctamers and longer probes will ligate substantially to completion (seebelow). In addition, adding a fluorescent moiety to the 5′ end of ahexamer probe seems to reduce the efficiency of ligation, whereas addinga fluorescent moiety to an octamer probe has little or no effect. Forthese reasons, use of octamers or longer probes is consideredpreferable.

Additional experiments (described below) have demonstrated ligation andcleavage of probes containing phosphorothiolate linkages anddegeneracy-reducing nucleotides; 3′ end specificity and selectivity ofligated extension probes; in-gel ligation and cleavage; sequentialcycles of primer hybridization and removal with minimal loss of signal;100% fidelity for T4 or Taq ligase for 3′→5′ extensions; and 4-colorspectral resolvability of ligated extension probes. An automated systemfor performing the methods has been constructed.

EXAMPLE 2 Efficient Cleavage and Ligation of PhosphorothiolatedOligonucleotides Containing Degeneracy-Reducing Nucleotides

A competing consideration to probe length, however, is the fidelity ofthe extended oligonucleotide and its effect on subsequent ligationefficiency. The fidelity of T4 DNA ligase has been shown to decreaserapidly following the 5^(th) base after the junction (Luo et al.,Nucleic Acid Res., 24: 3071-3078 and 3079-3085, 1996). If mismatches areintroduced at the 5′ side of a new ligation junction, the ligationefficiency may be reduced by attrition, however, no dephasing orincrease in background signal will be generated (a major obstacleencountered in polymerase-based sequencing by synthesis methods).

Probe sets should preferably be capable of hybridizing to any DNAsequence in order to permit de novo sequencing of uncharacterized DNA.However, the complexity of a labeled probe set grows exponentially withthe length and number of 4-fold degenerate bases. In addition, a complexprobe set is more challenging to synthesize while maintainingapproximately equal representation of all probe species, and is harderto purify. It also requires a higher concentration of probe mixture tomaintain a constant concentration of each species. One way to managethis complexity is to use nucleotides incorporating universal bases,such as deoxyinosine, at certain positions instead of 4-fold degeneratebases.

Twelve octanucleotide probes were designed with 4-fold degenerate bases(N; equimolar amounts of A, C, G, T) and the universal base inosine (I)at various positions within the octamer (inosine is capable ofbi-dentate hydrogen bonding with any of the four canonical bases inB-DNA; the order of stabilities of inosine base pairs isI:C>I:A>I:T≈I:G). One purpose for evaluating these probe designs was todetermine how low an octamer complexity could be achieved while stillsupporting efficient ligation in the presence of inosine bases.

In initial studies, several oligonucleotide probes were ligated tobead-based templates (long-LST1) using T4 DNA ligase. Upon ligation, thefluorophore-labeled primer (3′FAM Primer) shifts right in proportion tothe amount of oligonucleotide probe ligated. Probe design NI8-9 showedthe highest level of completion, with >99% of the primer populationshifting right due to efficient ligation of the probe (see FIG. 9).These reactions were conducted at 25° C.; when the reaction temperaturewas increased to 37° C., ligation was somewhat less efficient and thecompletion rates were more variable.

Closer examination of the data indicated that probes with fewer inosinebases within the first five nucleotides on the 3′ side of the junction(underlined) showed higher ligation efficiencies. To investigate furtherand to evaluate potential sequence context effects on ligationefficiencies, four oligonucleotide probe designs with only a singleinosine residue within the first five bases 3′ of the ligation junctionwere screened across all templates. FIG. 10 demonstrates ligationcompletion as evaluated using the gel-shift assay with selected probecompositions on multiple templates using T4 DNA ligase. Data from theseinitial experiments demonstrated that ligation efficiency, and hencecompletion, is variable and sequence-dependent when inosine residues areplaced within the first five 3′ positions of the ligation junction(underlined). Efficient ligation of octamers was observed consistently,however, with oligonucleotide probe design NI8-9, as demonstrated herewith >99% completion on all templates tested.

While not wishing to be bound by any theory, this data (including thepresence of adenylated intermediates) support the conclusion thatunfavorable inosine base pairs within the core DNA binding site for T4DNA ligase destabilize the DNA protein complex sufficiently to reduceenzyme binding and subsequent ligation. An interesting question,however, was whether such destabilizing inosine base pairs would affectthe fidelity of the ligated oligonucleotide probes.

EXAMPLE 3 Fidelity of Probe Ligation

Bacterial NAD-dependent ligases, such as Taq DNA ligase, have beenreported to have high sequence fidelity across ligation junctions, withmismatches on the 3′ side having essentially no nick-closure activity,but mismatches on the 5′ side being tolerated to some degree (Luo etal., Nucleic Acid Res., 24: 3071-3078 and 3079-3085, 1996). T4 DNAligase, on the other hand, has been reported to be somewhat lessstringent, allowing mismatches on both the 3′- and 5′-sides of thejunction. It was therefore of interest to evaluate the fidelity of probeligation with T4 DNA ligase in comparison to Taq DNA ligase in thecontext of our system.

We developed two methods to evaluate the sequence fidelity of ligatedoligonucleotides using standard ABI sequencing technology. The firstmethod was designed to clone and sequence ligation products. In thismethod, ligation extension products were attached to adapter sequences,cloned and transformed into bacteria. Individual colonies were pickedand sequenced to provide a quantitative assessment of the mismatchfrequency at each position across the ligation junction. The secondmethod was designed to sequence of ligation products directly. In thatapproach, single-stranded ligation products were denatured frombead-based templates and sequenced directly using a complementaryprimer. Positions with low accuracy display multiple overlapping peaksin the resulting sequence traces, providing a qualitative assessmentthat is indicative of the sequence fidelity at that position.

The first method was used to assess the relative fidelity of probeligation by T4 and Taq DNA ligases. A single bead-based templatepopulation (LST1) was hybridized to a universal sequencing primer, whichwas used as an initializing oligonucleotide. Solution-based ligationreactions were then performed in the presence of a degenerateoligonucleotide probe (N7A, 3′ANNNNNNN5′, 2000 pmoles) at 37° C. for 30minutes with either T4 DNA ligase (15 U per 1×10⁶ beads) or Taq DNAligase (60 U per 1×10⁶ beads) (FIG. 11, panel A). The ligation productswere cloned and sequenced to evaluate the positional fidelity of eachDNA ligase on the 3′ side of its ligation junction (Positions 1-8) (FIG.11, panels B and C). The results indicated that T4 DNA ligase hasessentially the same level of fidelity across the first 5 positions asTaq DNA ligase, but lower fidelity in positions 6-8. These results werefurther substantiated by subsequent cloning experiments that evaluatedDNA sequences across ligation junctions of all seven templates (LST1-7)for three degenerate, inosine-containing probe designs(3′-NNNNNIII-5′,3′-NNNNNINI-5′, and 3′-NNNINNNI-5′). The studiesconfirmed that T4 DNA ligase has low sequence fidelity across ligationjunctions at positions 6-8, however, high fidelity was exhibited acrossthe first 5 positions in all templates tested (data not shown).

The direct sequencing method was used to assess the fidelity of T4 DNAligase with degenerate, inosine-containing probes. Oligonucleotideprobes were evaluated at 25° C. and 37° C. in ligation reactions thatcontained T4 DNA ligase and bead-based templates. Oligonucleotide probeligation efficiencies were evaluated using a gel-shift assay (FIG. 12,panel A). Direct sequencing of the ligation reactions using an ABI3730x1DNA Analyzer was conducted to assess the fidelity of T4 DNA ligase inoligonucleotide probe ligation (FIG. 12, panel B). Ligation of an exactmatch oligo probe and two representative degenerate inosine-containingoligo probes (NI8-9 and NI8-11) gave >99% completion and a very lowfrequency of mismatches (absence of multiple peaks in the sequencingtraces). The data suggest that probes which are efficiently ligated alsogive high sequence fidelity.

In additional experiments, a single bead-based template population(LST1) was hybridized to a universal sequencing primer that contained 5′phosphates, which was used as an initializing oligonucleotide.Solution-based ligation reactions were performed at 37 C for 30 minuteswith T4 DNA ligase (1 U per 250,000 beads) in the presence of adegenerate, inosine-containing oligonucleotide probe (3′NNNNNiii5′,3′NNNNNiNi5′, or 3′NNNiNNNi5′, 600 pmoles). Ligation products werecloned and colonies were picked and sequenced. Sequence fidelity wasdetermined by calculating the number of clones represented for eachposition across the ligation junction. Results are tabulated in FIG. 12,panels C-F. These studies demonstrate that 3′→5′ ligation of degenerate,inosine-containing probes with T4 DNA ligase has high-level fidelity inthe first 1-5 positions.

EXAMPLE 4 In-Gel Ligation and Cleavage

The initial experiments to explore, develop and optimize methods forcycled oligonucleotide ligation were conducted using bead-basedtemplates in solution, as described above. In a second set ofexperiments, ligation and cleavage were performed on bead-basedtemplates that were embedded in polyacrylamide gels on slides.

Slides were prepared by mixing millions of beads, each having a clonalpopulation of single-stranded DNA templates attached thereto, with 5%polyacrylamide and allowing polymerization to occur on a glass slide. ATeflon® mask was used to enclose the bead-containing polyacrylamidesolution. FIG. 14 (top) shows a fluorescence image of a portion of aslide on which beads with an attached template, to which a Cy3-labeledprimer was hybridized, were immobilized within a polyacrylamide gel.(This slide was used in a different experiment, but is representative ofthe slides used here.) FIG. 14 (bottom) shows a schematic diagram of aslide equipped with a Teflon mask to enclose the polyacrylamidesolution.

Reactants were introduced into slides either by manual dipping of slidesinto appropriate solutions or by placing the slides in an automated,laminar flow cell. Initial studies confirmed that efficient in-gelligation could indeed be performed on templates attached to beadsimmobilized in a polyacrylamide matrix on such slides. In the experimentshown in FIG. 15, single-stranded DNA template beads were immobilized onslides containing acrylamide and DATD. Following polymerization, auniversal, 3′ fluorophore-labeled, 5′ phosphorylated primer (Seq Primer)was diffused into the gel and allowed to hybridize (panel A). Slideswere washed to remove unbound seq primer, overlaid with a ligationcocktail that contained T4 DNA ligase (10 U) and an oligonucleotideprobe, and incubated at 37° C. for 30 minutes. Slides were thenincubated in a buffer containing sodium periodate (0.1M) to digest theacrylamide polymer and to release the bead-based template populations.Ligated products were denatured from the template strand by heat,collected and analyzed using the gel shift assay described above. In-gelligation reactions performed in the absence of T4 DNA ligasedemonstrated a single peak representative of unligated sequencing primer(panel B). Ligation reactions performed with octamer probes in thepresence of T4 DNA ligase demonstrated efficient in gel oligonucleotideligation with >99% of bead-based template populations efficientlyligated (panel C).

EXAMPLE 5 Four-Color Detection

To maximize detection efficiency, it is desirable to employ a set ofoligonucleotide probes with distinct labels corresponding to eachpossible base addition product. This was modeled in our automatedsequencing instrument equipped with appropriate excitation and emissionfilters, as outlined in FIG. 15. Three sets of octamer probes weredesigned to address issues of probe specificity and selectivity. Thefirst set included four octamers, complementary to four unique templatepopulations, with different 3′ bases and 5′ dye labels. The second setincluded seven unique octamers with unique 3′ bases and 5′ dyes. Thethird set corresponded to a probe design with four degenerate,inosine-containing octamers, each having a unique 3′ end base identifiedby a different 5′ dye label.

To confirm four-color spectral identity, probe set #1 was employed todetect four unique template populations (see FIG. 16). Slides wereprepared containing four, unique single-stranded template populationsattached to beads, which were embedded in polyacrylamide (panel A). Eachbead had a clonal population of templates attached thereto. A universalsequencing primer containing 5′ phosphates was hybridized, in situ, andligation reactions were performed using an oligonucleotide probe mixturethat contained four unique fluorophore probes (Cy5, CAL 610, CAL 560,FAM; 100 pmoles each) and T4 DNA ligase (10 U/slide). Slides wereincubated at 37° C. for 30 minutes and washed to remove unbound probes.The slides were imaged in bright light to create a white light baseimage (panel B) and with fluorescence excitation using the four bandpassfilters (FITC, Cy3, TxRed, and Cy5). Fluorescence image capture wasconducted pre- and post-ligation. Individual populations werepseudocolored (panel C) and the spectral identity of image values wereplotted and confirm minimal signal overlap (panel D).

EXAMPLE 6 Demonstration of Ligation Specificity and Selectivity in Gels

To confirm 3′ end specificity, probe set #2, was used to interrogate asingle template population (see FIG. 17). Slides were prepared with abeads having a single template population (LST1.T) attached theretoembedded in a polyacrylamide gel, and were hybridized, in situ, with auniversal sequencing primer (panel A). In-gel ligation reactions wereconducted with T4 DNA ligase (10 U/slide) using an oligonucleotide probemixture comprised of four 5′ end-labeled probes that differed only by asingle 3′ base. Slides were incubated at 37° C. for 30 minutes andwashed to remove unbound probe populations. Slides were imaged in whitelight to create a base image (panel B) and with fluorescence excitationusing four bandpass filters (FITC, Cy3, TxRed, and Cy5). Fluorescenceimage capture conducted pre- and post-ligation confirmed a singleFAM-based probe population (blue spots) present following in-gelligation with T4 DNA ligase, with no spectral overlap (panels C, D).This data demonstrates that probe specificity with T4 DNA ligase isstringent and is determined by the first 3′ end base of the ligationjunction.

To further substantiate 3′ end specificity and selectivity, probe set #2was used to identify a mixture of bead-based template populationscontaining single base differences and present in different amounts.Slides were prepared with mixtures of beads each having one of fourtemplate populations, each with a single nucleotide polymorphism (LST1;A, G, C or T), attached thereto, as indicated in panel A of FIG. 18. Thebeads were embedded in a polyacrylamide gel on the slide. Bead-basedtemplate populations were used at various different frequencies, asoutlined in panel D. Slides were hybridized, in situ, with universalsequencing primers. In-gel ligation reactions were conducted using T4DNA ligase (10 U/slide) and an oligonucleotide probe mixture containingequimolar amounts (100 pmoles, each) of four 5′ end-labeled probes thatdiffered only by a single 3′ base. Slides were incubated at 37° C. for30 minutes and washed to remove unbound probe populations. Slides wereimaged in white light to create a base image (panel B) and withfluorescence using four distinct bandpass filters (FITC, Cy3, TxRed, andCy5). Individual probe images were overlaid and pseudocolored (panel C).Fluorescent images were enumerated using bead-calling software. Theresults are presented in panel D and confirm that observed ligationfrequencies (Obs) correlated with the expected frequencies (Exp). Thedata demonstrate high probe specificity and probe selectivity afterligation in the presence of multiple templates and demonstrate thecapability of detecting single nucleotide polymorphisms (SNPs), i.e.,alterations that occur in a single nucleotide base in a stretch ofgenomic DNA in different individuals of a population, by ligation.

EXAMPLE 7 Demonstration of Ligation Specificity and Selectivity in GelsUsing Four-Color Degenerate Inosine-Containing Extension Probes

Another set of experiments were conducted, using probe set #3, toevaluate the specificity and selectivity of probe ligation usingfour-color degenerate, inosine-containing oligonucleotide probe pools.Results are presented in FIG. 19. Bead-based slides were prepared asdescribed above, but with four, unique single-stranded templatepopulations present on beads in different amounts and were thenhybridized, in situ, with a universal sequencing primer (panel A).In-gel ligation reactions were performed in the presence of T4 DNAligase (10 U/slide) using probe pools consisting of octamers designedwith five degenerate bases (N; complexity 4⁵=1024), two universal bases(I, inosine), and single known nucleotide at the 3′ end corresponding toa specific 5′ fluorophore (G-Cy5, A-CAL 610, T-CAL560, A-FAM; 600 pmoleseach). Slides were incubated at 37° C. for 30 minutes and washed toremove unbound probe populations. Slides were imaged in white light tocreate a base image (panel B) and with fluorescence using four bandpassfilters (FITC, Cy3, TxRed, and Cy5). Individual probe images wereoverlaid and pseudocolored (panel C). Fluorescent images were enumeratedand the frequencies of each ligation product tabulated usingbead-calling software (panel D); spectral scatter plots of unprocessedraw data and filtered data representing the top 90% of bead signalvalues are shown in panel E. The data demonstrate that the observedligation frequencies (Obs) correlated with the expected frequencies(Exp) based on the known concentrations of each template. This confirmsthat degenerate and universal base-containing probe pools can be usedwith T4 DNA ligase to afford specific and selective in-gel ligation.

EXAMPLE 8 Demonstration of Repeated Cycles of Hybridization and Removalof Initializing Oligonucleotide in Gel

Experiments conducted on templates immobilized in a gel on a microscopeslide mounted in an automated flow cell (see below) confirmed thatmultiple cycles of annealing and stripping an initializingoligonucleotide could be applied to templates attached to beads embeddedin gels on slides with minimal signal loss. A 44 base fluorescentlylabeled initializing oligonucleotide was used. As shown in FIG. 20,minimal signal loss occurred over 10 cycles. The initializingoligonucleotide is referred to as a primer in FIG. 20. As indicatedabove, one of the major drawbacks of polymerase basedsequencing-by-synthesis procedures is the propensity for both positiveand negative dephasing to occur on individual template strands. Positivedephasing occurs when nucleotides are misincorporated in a growingstrand, hence causing the base sequence of that particular strand to runahead of the sequence obtained from the remaining templates and to beout of phase by n+1 base calls. Negative dephasing, which is morecommon, occurs when strands are not fully extended, resulting inbackground base calls that run behind the growing strand (n−1). Theability to efficiently strip extension products and to “reset” templatesby hybridizing a differentially positioned initializing oligonucleotideallows very long read lengths with little to no signal attrition.

EXAMPLE 9 Automated Sequencing System

This example describes a representative inventive automated sequencingsystem that can be used to gather sequence information from one or moretemplates. Preferably the templates are located on a substantiallyplanar substrate such as a glass microscope slide. For example, thetemplates may be attached to beads that are arrayed on the substrate. Aphotograph of the system is presented in FIG. 21. The system is based onan Olympus epi-fluorescence microscope body (mounted sideways) with anautomated, auto-focusing stage and CCD camera. Four filter cubes in arotating holder permit four-color detection at a variety of excitationand emission wavelengths. A flow cell with peltier temperature control,which can be opened and closed to accept a substrate such as a slide(with a gasket to seal around the edge of an area containing asemi-solid support such as a gel), is mounted on the stage. The verticalorientation of the flow cell is an important aspect of the inventivesystem and allows air bubbles to escape from the top of the flow cell.The cell can be completely filled with air to eject all reagents priorto each wash step. The flow cell is connected to a fluid handler withtwo 9-port Cavro syringe pumps, which allow delivery of 4 differentiallylabeled probe mixtures, cleavage reagent, any other desired reagents,enzyme equilibration buffer, wash buffer and air to the flow cellthrough a single port. The operation of the system is completelyautomated and programmable through control software using a dedicatedcomputer with multiple I/O ports. The Cooke Sensicam camera incorporatesa 1.3 megapixel cooled CCD though cameras having lesser or greatersensitivity could also be used (e.g., 4 megapixel, 8 megapixel, etc.,can be used). The flow cell utilizes a 0.25 micron stage, with a 1micron feature size.

EXAMPLE 10 Image Acquisition and Processing Methods

This example describes representative methods for acquiring andprocessing images from arrays of beads having labeled nucleic acidsattached thereto. Accurate feature identification and alignment areimportant for reliable analysis of each acquired image. The features areidentified by first discarding all but the most intense pixels for eachbead. The pixel values for a given image are plotted in a histogram;pixels corresponding to background are discarded and the remaining pixelvalues are sorted. In uniform images, where all the beads are roughlythe same intensity, the algorithm eliminates the bottom 80-90% of pixelvalues. Pixels having values in the top 10-20% are then scanned toidentify those at a local maximum in a 4 pixel radius. The averageintensity in that region as well as the average intensity of theperimeter are then recorded. These values form a normal distribution andpixels whose values fall outside that distribution are then removed. Thepercentage of pixels initially ignored, the size of the circular region,and the cutoff values that eliminate possible beads in the normaldistribution are all parameterized and can be tuned if necessary.Alignment is accomplished by creating feature matrices for each image inthe alignment set. The resulting matrices are then searched for the mostfrequent x,y coordinate offsets to identify the optimal alignment.

Bead images are collected in the Cy5 channel (corresponding to thesequencing primer) prior to extension probe addition. These images areused to create a feature map marking both positional coordinates and rawsignal intensities as fluorescent units (RFU values) for each bead. Foreach subsequent duplex extension, an image set is acquired both beforeand after the Cy3-labeled nucleotides are added. These images arealigned to the original Cy5 images and RFU values are then assigned toeach of the beads and recorded. A baseline correction is applied bysubtracting the difference of intensities between the unlabeled(pre-extension) and labeled (fluorescent-addition) images of each baseaddition. These baseline-subtracted values are then normalized by theintensity found in the Cy5 image for each feature to form the basis bywhich a bead is considered to have been extended or not (i.e., a bead isconsidered to be extended if duplexes attached to the bead wereextended). Using these methods thousands of features per image with˜1,300 images per slide can be analyzed to afford an analysis of 5-100million template species per experimental run. The algorithms have beendesigned so that they can be easily ported from MATLAB to C+ at a laterdate for further efficiency enhancements.

EXAMPLE 11 Bead Alignment and Tracking and Sequence Decoding

This example describes representative methods for processing images fromarrays of beads having labeled nucleic acids attached thereto and forsequence determination from the acquired data.

Image analysis starts by convolving the image using a zero-integralcircular top-hat kernel with a diameter matched to the bead size. Thiswill automatically normalize the background to zero while identifyingthe centers of individual beads through local maxima. The maxima arelocated and those which are isolated from other local maxima are used asalignment points. These alignment points are computed for each image ina time-series. For each pair of images, the alignment points arecompared and a displacement vector is computed based on the averagedisplacement of all the common alignment points. This provides pair-wiseimage displacements with sub-pixel resolution.

For N images, there are N*(N−1)/2 pairwise displacements, but only N−1of these are independent since the rest can be calculated from theindependent set. For example, measuring the displacements between images1 and 2 and between images 1 and 3 implies a displacement between images2 and 3. If the measured displacement between images 2 and 3 is not thesame as the implied displacement, then the measurements areinconsistent. The magnitude of this inconsistency can be used as ametric to gauge how well the alignment algorithm is working. Our initialtests show inconsistencies that are generally less than 0.1 pixel ineach dimension (see FIG. 23).

Once a time-series of images is aligned, there are two ways to track theindividual beads. If the bead density is low with most of the beads nottouching another bead, the optical center-of-mass of each individualbead can be identified and a region around the bead integrated tocompute the bead intensity. If the bead density is so high that most ofthe beads touch, then it is not possible to identify individual beads bya dark background band around them. However, with all the images alignedto sub-pixel resolution, it is possible to identify pixels belonging tothe same bead by computing the correlation, in time, of adjacent pixels.Highly correlated pixel pairs can be confidently assigned to the samebead. A similar technique has been applied to lane tracking in DNAsequencing gels with good results (Blanchard, A. P. Sequence-specificeffects on the incorporation of dideoxynucleotides by a modified T7polymerase, California Institute of Technology, 1993). Once the beadshave been tracked through the entire 4-color time-series, the sequenceis decoded by knowing which color corresponds to which 3′-most base ofthe probe oligonucleotides.

EXAMPLE 11 Throughput Calculations

In general, the throughput of the sequencing system is defined primarilyby the number of images that the machine can generate per day and thenumber of nucleotides (bases) of sequence data per image. Since themachine is preferably designed to keep the cameras constantly busy,calculations are based on 100% camera utilization. In implementations inwhich each bead is imaged in 4 colors to determine the identity of onebase, either 4 images by one camera, 2 images by 2 cameras, or one imageby 4 cameras can be used. Four-camera imaging permits dramaticallyhigher throughputs than the other options, and preferred systems utilizethat approach.

Our initial tests show that a pixel density of 50 pixels per bead,representing 5.4 square microns, provides a comfortable density forstandard image analysis. By using a 4 megapixel CCD camera (nowcommonplace), a single CCD frame can image ˜80,000 beads (based on ourcurrent image data). Capturing four images with separate cameras andmoving to the next field on the flow cell will take no longer than 1.5seconds. If 75% of the beads yield useful information, we will be ableto collect data from approximately 80,000 beads*0.75/1.5=40,000bases/sec of raw sequence data.

One significant issue in maintaining 100% camera utilization is matchingthe time it takes to perform one cycle of ligation/cleavage chemistrywith the time required to image the entire flow cell. A reasonableestimate for the time taken by a cycle of extension, cleavage, andligation is 1½ hours (5,400 seconds). That 5,400 seconds willaccommodate 1,800 image fields, or an area of about 15 mm×45 mm, whichis a comfortable size for a flow cell. A conservative estimate of thethroughput of the system utilizing four cameras is 40,000 bases persecond with a 15 mm×45 mm flow cell. This is equivalent to approximately2,000 ABI3730x1 sequencing machines, based on a throughput of 28 runsper day with ˜650 base read lengths (20 bases/sec), which we haveachieved using these machines. A 2.5 fold increase in bead density, to200,000 per image enables an overall increase in throughput to 100,000bases per second, approximately equivalent to 5,000 ABI3730x1 machines.The total output per day at this throughput level is ˜8.6 Gb per day, sothe time required to complete a 12× human genome sequence would be ˜4.2days.

It is noted that the inventive sequencing methods described herein maybe practiced using a variety of different sequencing systems, imagecapture and processing methods, etc. See, e.g., U.S. Pat. Nos. 6,406,848and 6,654,505 and PCT Pub. No. WO98053300 for discussion.

EXAMPLE 12 Methods for Preparing Microparticles for Template SynthesisThereon

This example describes a protocol preparation of microparticles (in thisexample, magnetic beads) with amplification primers attached thereto sothat a template can be amplified (e.g., by PCR) so as to result in aclonal population of template molecules attached to each microparticle.In general, amplification beads have one primer needed in the clonal PCRreaction attached thereto. This primer can be covalently coupled or, forexample, biotin labeled and bound to streptavidin on the bead surface.Beads can be used in a standard PCR reaction (e.g., in wells of amicrotiter plate, tubes, etc.), in an emulsion PCR reaction as describedin Example 13, etc., to obtain beads having clonal populations oftemplate molecules attached thereto.

Materials

-   1× TE: 10 mM Tris (pH 8) 1 mM EDTA-   1×PCR buffer: (ThermoPol Buffer, NEB)-   20 mM Tris-HCl (pH 8.8)-   10 mM KCl-   mM (NH₄)₂SO₄-   2 mM MgSO₄-   0.1% Triton X-100-   1M Betaine (add only for 1×PCR-B buffer)-   1× Bind & Wash Buffer-   5 mM Tris HCl (pH 7.5)-   0.5 mM EDTA-   1 M NaCl-   DNA Capture Primer (20-mer, 500 μM stock)

Dual Biotin-(HEG)5-P1: (SEQ ID NO. 4)5′-Dual Biotin-(HEG)5-CTA AGG TAG CGA CTG TCC TA-3′(HEG)5=Hexaethylene glycol linker, an 18 carbon containing spacer, oneof a number of different spacer moieties that could be used. Including aspacer is useful, e.g., to raise the P1 primer portion of the oligo offthe surface of the bead. Any of the primers described herein mayincorporate such spacer moieties.Dynal stock magnetic beads (1 μm diameter)=10 mg/ml (7−12×10⁶ beads/μl).

Methods

-   1. Remove 50 μl beads (˜450×10⁶ beads).-   2. Add 200 μl 1×TE buffer, mix well. Separate with magnet.-   3. Wash 1× with 200 μl 1×TE buffer. Separate with magnet.-   4. Resuspend in 100 μl B/W buffer.-   5. Add 3 μl of P1 oligo (500 μM stock=1500 μmol).-   6. Rotate at RT for >30 minutes.-   7. Wash 3× with 200 μl 1×TE buffer.-   8. Resuspend in 500 (initial volume) 1×TE buffer.-   9. Store DNA capture beads at 4 C or place on ice prior to use.    Beads should be used within 1 week (beads will tend to clump at    storage times >1 week).

EXAMPLE 13 Methods for Performing PCR on Microparticles in an Emulsion

This example describes methods that can be used to perform PCR onmicroparticles in an emulsion to produce microparticles with clonaltemplates attached thereto. The microparticles (DNA beads in thenomenclature used below) are first functionalized with a first primer(P1). A second primer (P2) is present in the aqueous phase, where thePCR reaction occurs. If desired, a low concentration of P1 may also beincluded, e.g., (20-fold less) in the aqueous phase. Doing so allows arapid build-up of templates in the aqueous phase, which are substratesfor additional amplification. As P1 is depleted in solution, thereaction is driven towards utilization of P1 attached to themicroparticles. P1_P2 degen10 is an oligonucleotide template (100 bp)that has sequences that hybridize to P1 and P2 to afford amplificationby PCR and a stretch of approx 10 degenerate bases (incorporated duringoligonucleotide synthesis) that give the oligonucleotide population acomplexity of 4¹⁰

I. Emulsion Protocol (1 μm beads)

-   1. Prepare oil phase:

Span 80 (7%)

Tween 80 (0.4%)

Prepared in Light Mineral Oil

Use only freshly made oil phase

Total Oil Phase=450 μl

-   2. Prepare aqueous phase: (Estimated to produce 2×10⁹ droplets, 115    fL per droplet)

Reagent (stock) (μl) per reaction Final dH₂O 156.0 — MgCl₂ Buffer (10X)32.0 1X dNTP (100 mM ea) 11.3 3.5 mM each MgCl₂ (1M) 7.3 23 mM Betaine(5M) 32.0 0.5M P1 (Primer 1) (10 μM) 1.6 11.25 pmole P2 (Primer 2) (200μM) 40.0 5625 pmole P1_P2 degen10 (100 pM) 6.6 5.9 × 10{circumflex over( )}7/ul DNA Beads (8 M/μl) 25.0 150M/emulsion Platinum Taq (5 U/μl) 9.00.28 U/ul Total aqueous volume = 320 μl Final reaction = 255 μl aqueousphase:450 μl oil phase

-   -   3. Transfer aqueous phase tube to ice until addition to        emulsion.    -   4. Add 450 μl oil phase to a 2 ml cryovial.    -   5. Place cryovial UPRIGHT into foam adapter attached to IKA        vortex. Set vortex to 2500 rpm.    -   6. Aliquot aqueous phase (3 aliquots, 85 μl each=255 μl) to        shaking oil phase. Add monodispersed aqueous phase to the        agitating 2 ml cryovial by placing the tip into tube and slowly        dispensing the aqueous phase from the tip into the shaking oil        phase. Repeat addition 2× with the remaining aqueous phase.    -   7. Continue shaking emulsion for 24 minutes at 2500 rpm.    -   8. Transfer ˜100 μl aliquots of the emulsion into a 96-well        plate (total=4 wells). Also, aliquot remaining aqueous phase (65        μl) into a separate well for a solution-based PCR control        reaction. Seal plate and cycle as outlined in next section.        II. Emulsion Amplification (1 μm beads)

-   1. PCR cycling parameters for 1 μm bead emulsions (with primer    Tms=62 C):

Program: DTB-PCR

94 C, 2 min n=1

94 C, 15 s

57 C, 30 s n=100

70 C, 60 s

55 C, 5 min n=1

10 C, for arbitrary time period

-   2. Cycling time is ˜6 hours.-   3. Observe emulsions following cycling. Successful emulsions will    appear uniformly amber in color with no observable separated aqueous    phase. Emulsions that “break” (fall out of solution) will have a    distinct aqueous phase at the bottom of the tube. Avoid collecting    this phase, as this population of beads will not be clonal.-   4. Assess post-cycled emulsions using bright field microscopy.    Remove a 2 μl aliquot of the cycled emulsion and drop onto a glass    slide. Overlay emulsion sample with a 22×60 mm glass coverslip.-   5. View emulsions using the 20× objective. Beads should preferably    be monodispersed, with the majority of droplets containing single    beads.    NOTE: If the emulsion sample contains a high number of multi-bead    droplets, pool emulsion reactions into a single 1.5 ml eppendorf    tube and spin at 6000 rpm for 15 seconds. Remove the bead suspension    that accumulates at bottom of tube. This population will be    comprised of both free beads and multi-bead droplets that are    heavier than single-bead droplets and thus will settle to the bottom    of the tube following a brief spin. This bead population is not    clonal and should therefore be avoided prior to subsequent    processing. Re-evaluate emulsion by repeating Steps 4 and 5 to    confirm integrity of single bead-containing droplets in emulsion    sample.-   6. Disrupt (break) emulsions using the protocol outlined in the next    section.-   III. Emulsion Break and Melt (1 μm beads)-   Bead Break Wash (BBW) Buffer-   2% Triton X-100 2% Tween 20; 10 mM EDTA-   Melt Solution 100 mM NaOH-   1×TE: 10 mM Tris (pH 8) 1 mM EDTA-   1× Bind & Wash (B/W) Buffer-   5 mM Tris-HCl (pH 7.5)-   0.5 mM EDTA-   1 M NaCl-   1. Pool each emulsion set (4 aliquots) into a single 1.5 ml    eppendorf tube.-   2. Add 800 μl BBW buffer. Break emulsions by vortexing reaction tube    for 10 seconds.-   3. Spin at 8000 rpm for 2 min.-   4. Remove top 800 μl (mainly oil phase). DNA beads will be pelleted    at the bottom of tube.-   5. Add 800 μl BBW, vortex and spin at 8000 rpm for 2 min. Remove top    600 μl.-   6. Wash an additional 2× with 600 μl 1×TE using a magnet to exchange    each wash.-   8. Add 50 μl Melt solution to bead pellet and resuspend sample by    vigorous pipetting. Incubate beads in Melt solution for 5 minutes at    room temperature, flicking tube intermittently.-   9. Place tube in magnet to remove Melt solution. Wash 1× with 100 μl    Melt solution to ensure complete removal of second strand.-   10. Wash bead pellet 2× with 1×TE and resuspend into 20 μl TE buffer    for storage at 4 C or 20 μl 1× B/W buffer if next step is    enrichment. If beads appear to be clumped, exchange into 1×PCR-B    buffer.-   11. Continue with enrichment protocol (optional).

EXAMPLE 14 Methods for Enriching for Microparticles Having ClonalTemplate Populations Attached Thereto

This example describes a method for enriching for microparticles onwhich template amplification has successfully occurred in, e.g, in a PCRemulsion. The method makes use of larger microparticles that have acapture oligonucleotide attached thereto. The capture oligonucleotidecomprises a nucleotide region that is complementary to a nucleotideregion present in the templates.

I. Emulsion Enrichment (1 μm)

A. Preparation of Enrichment Beads (Capture Entities)

-   -   Enrichment Beads:    -   Spherotech streptavidin-coated polystyrene beads (˜6.5 um)    -   Bead stock (0.5% w/v): 33,125 beads/μl    -   Per Protocol: (33,125 beads/μl) (800 μl)=26.5×10⁶ beads    -   Usage:    -   119 million beads per emulsion—estimate of emulsion clonality        (2%): ˜3M template-positive beads per emulsion. Add 2-3        enrichment beads per estimated template-positive emulsion        bead=10 million enrichment beads per emulsion reaction.        Enrichment Oligonucleotide (Capture Agent):

-   P2-enrich (35-mer, Tm=73 C)

-   5′-Dual biotin-18-carbon spacer-ttaggaccgttatagttaggtgatgcattaccctg    3′(SEQ ID NO. 5) (or)    P2-enrich (e.g., up to 35-mer, Tm=52 C)

-   5′-Dual Biotin-18-carbon spacer-ggtgatgcattaccctg 3′ (SEQ ID NO. 6)    Glycerol Solution—60% (v/v)    -   6 ml glycerol    -   4 ml nuclease-free H₂O

-   1. Remove 800 μl of beads and exchange into B/W buffer by    centrifugation at 13,000 rpm for 1 minute. Wash 1× with 500 μl B/W    buffer and resuspend into 100 μl B/W buffer.

-   2. Add 20 μl enrichment oligo (500 μM stock=10,000 pmoles per r×n).

-   3. Rotate bead reaction at room temperature for 1 hour.

-   4. Wash beads 3× using 500 μl 1×TE buffer. Pellet beads between    washes by centrifugation at 13,000 rpm for 1 minute.

-   5. Resuspend beads into 25 μl B/W buffer. Concentration=1M    enrichment beads/μl.    NOTE: Pooling four enriched emulsion populations into 20-30 μl 1×    B/W buffer yields 40M template-positive beads. Multiple slides can    then be run.    B. Enrichment Procedure

-   1. Add 20 μl of the enrichment beads to the tube containing    emulsion-derived beads (20 μl). Resuspend bead mixture with gentle    pipetting (or use ratios that give rise to 2-3 enrichment beads for    every estimated template-positive emulsion bead).

-   2. If using enrichment beads coated with the biotinylated P2-enrich    primer, incubate bead mixture at 65 C for 2 minutes. Remove tube to    ice for 10 minutes.    NOTE: Initial experiments have suggested that using enrichment beads    containing primer sequences used for the 100-cycle PCR (e.g., P2PCR)    may be less efficient at enrichment due to the ability to enrich for    beads containing primer:dimer species driven to bead in droplets    that were devoid of template. If using enrichment beads loaded with    the P2-enrich primer described above, incubate bead mixture at 50 C    for 2 minutes due to the reduced Tm of this shorter primer.

-   3. Overlay bead mixture into 1.5 ml eppendorf tube containing 300 μl    60% Glycerol solution.

-   4. Centrifuge at 13,000 rpm for 1 minute.

-   5. Following spin, negative beads will pellet to bottom of tube.    Enrichment beads containing attached template beads will float to    the top of the glycerol phase. Collect top-phase bead population and    transfer to a clean 1.5 ml eppendorf tube.    NOTE: Beads pelleted to the bottom of the tube (beads with no    template) can be washed and analyzed using a magnet following the    same wash regimen as outlined for template-positive beads.

-   6. To beads pulled from top phase, add 1 ml nuclease-free H2O to    dilute the glycerol concentration. Resuspend bead mixture using    gentle pipetting. Spin at 13,000 rpm for 1 minute.

-   7. Following spin, remove supernatant and wash 2× using 100 μl TE.

-   8. Add 100 μl Melt solution to the washed bead pellet. Rotate tube    for 5 minutes at room temperature.

-   9. Add an additional 100 μl Melt solution and isolate template beads    using a magnet.

-   10. Remove non-magnetic enrichment beads by washing 2× using 100 μl    TE and a magnet to pull DNA beads away from enrichment beads.

-   11. Resuspend template beads into 10-20 μl 1×TE. If beads appear to    be clumped, dilute into 1×PCR-B buffer.

-   12. Template-containing beads can be pooled with other enriched    populations and loaded onto slides as described in the next Example.

EXAMPLE 15 Methods for Preparing a Microparticle Array Immobilized in oron a Semi-Solid Support

This example describes preparation of slides on which microparticleshaving templates attached thereto are immobilized (e.g., embedded) in asemi-solid support located on the slide. Such slides may be referred toas polony slides. The semi-solid support used in this example ispolyacrylamide. One of the protocols employs methods that trappolymerase molecules in the vicinity of templates to enhanceamplification.

Preparation of Slides

A. Glass Slides: Bind-Silane Treatment

-   Bind-Silane facilitates the attachment of the acrylamide gel to the    glass slide surface.-   Slides should be pre-treated with Bind-Silane prior to use.    NOTES:    -   Store Bind-Silane solution in chemical hood.    -   Bind-Silane is an irritant. Work in a chemical when preparing        solution.    -   Ensure that the stock Bind-Silane solution has not expired.    -   Try not to touch surfaces of slides while transferring to and        from racks. Prepare Bind-Silane Solution:        -   1. In a 1-L plastic container add:        -   1 L dH2O, 1 Stir bar        -   Add 220 ul concentrated Acetic Acid (to generate pH 3.5) Add            4 ml Bind-Silane reagent Mix solution for >15 minutes using            stir plate.            Treat Slides:-   2. Load slides (facing the same direction) into upside-down plastic    384-well plates.-   3. Wash slides by rinsing with dH₂O, drain well.-   4. Rinse with 100% ethanol, drain well.-   5. Rinse again with dH₂O, drain well and place in tissue culture    hood with vent and UV light running. Allow washed slides to dry (˜30    min).-   6. Place plate into a plastic container and cover slides with    Bind-Silane solution.-   7. Allow solution and slides to react for 1 hour. Agitate container    intermittently to ensure even coating of Bind-Silane to glass.-   8. Following incubation, rinse slides 3× with dH2O.-   9. Rinse 1× with 100% ethanol, drain well.-   10. Allow slides to dry thoroughly prior to use.-   11. Store Bind-Silane-treated slides in dessicator.    B. Acrylamide-Based Slides (Small Mask)

Non-Trapping Protocol

-   1. Place all reagents on ice. Add the following chilled reagents to    a 1.5 ml eppendorf tube:

amt (μl) Reagent 2 slides 1 slide 1x TE 13 6.5 Beads (1-3M, diluted in1x TE) 10 5 Rhinohide 1 0.5 40% Acrylamide:Bis (19:1, F/S) 5 2.5 TEMED(5%, in 1x TE) 2 1 APS (0.5%, made fresh) 3 1.5 Total 34 μl 17 μl

Pipet mixture vigorously to distribute beads.

-   -   Load 17 μl per slide under a glass coverslip.    -   Polymerize upside down at room temperature for 60 minutes.    -   Remove coverslip with a clean razorblade.    -   Soak slide and wash 2× in 1E buffer for 15 minutes (to remove        unbound beads).    -   Slides with embedded beads can be stored at 4 C in wash 1E.

-   2. Hybridize fluorophore-labeled sequencing primer to embedded bead    population. Equilibrate slide from wash 1E to 1×PCR-B buffer by    dipping briefly into Coplin jar containing 1×PCR-B buffer.

-   3. In a 1.5 ml eppendort tube, add 1-6 μl (100 μM stock) primer to    99 μl 1×PCR buffer. Over the acrylamide matrix, drop 100 μl primer    solution and overlay with a glass coverslip or sealing gasket.

-   4. Hybridize primer to embedded beads by heating slide using <DEVIN>    program (65 C for 2 minutes, slow anneal to 30 C). Wash slide 2× for    2 minutes in wash 1E. Slide is ready to be subjected to ligation    based sequencing.    Trapping Protocol

-   1. ssDNA template beads are prepared at 1M/μl [Prepare polony slides    with 4-5M beads per slide].

-   2. Resuspend bead mixture into 30 μl×PCR buffer.

-   3. Add 1 ul sequencing primer (100 μM stock); mix well.

-   4. Heat to 65 C for 2 min.

-   5. Remove to ice for 5 min.

-   6. Wash 3× with 80 μl×TE

-   7. Remove all soln using a magnet.

-   8. Add reagents as outlined below:

amt(μl) Reagent 2slides 1x buffer 1.5 10x buffer 2.0 High conc.(HC)enzyme 16.0 40% Acrylamide:Bis (19:1, F/S) 14.4 Rhinohide 2.0 TEMED (5%,in 1x TE) 2.0 APS (0.5%, made fresh) 1.5 Total 39.4 μl

Pipet mixture to distribute beads.

Load 17 μl per slide under a glass coverslip.

-   9. Polymerize, preferably upside down, e.g., using <Pol-1> cycling    profile on MJ Research Tetrad PCR machine.-   10. Remove coverslip with a clean razorblade. Soak slide and wash 2×    in 1E buffer for 10 min. (to remove unbound beads).-   11. Polony slides are ready to be subjected to ligation-based    sequencing.-   12. Polony slides with embedded beads can be stored in gaskets at 4    C in wash 1E.

EXAMPLE 16 Methods for Preparing a Microparticle Array Attached to aSolid Support

This example describes preparation of slides on which microparticleshaving templates attached thereto are attached to a solid support.

1. Glass slides prepared with polymer tethers with reactive NHS arestored at −20 C. (Slide H, Product No. 1070936; Schott Nexterion; SchottNorth America, Inc., Elmsford, N.Y.)

2. In the presence of dessicant, equilibrate slides to room temperaturebefore use.

3. Wash slides in 50 mls 1×PBS (300 mM sodium phosphate, pH 8.7) for 5minutes. Repeat washes 2×.

4. Remove slide from solution and cover with an adhesive gasket (toallow sample loading).

5. In a separate tube, aliquot 100-400 million protein-coated orDNA-coated beads into 1×PBS, pH 8.7. The DNA can be, e.g., DNA templatesfor sequencing. The DNA can include, e.g., an amine linker for reactionwith NHS.

6. Wash bead sample 3× with 1×PBS, pH 8.7 by buffer exchange.

7. Resuspend beads into 125 ml 1×PBS, pH 8.7.

8. Load bead solution into the slide gasket to evenly coat slidesurface.

9. Enclose slides in a dark chamber and allow reaction to incubate for1-2 hrs at room temperature.

10. Following incubation, remove unbound bead solution and transferslide to 50 mls 1×TE (10 mM Tris, 1 mM EDTA, pH 8).

11. Wash slide 5× using 50 mls 1×TE with constant agitation for 15minutes per wash.

12. Slides can be stored in 1×TE at 4 C for several weeks.

13. If desired, bead populations can be assessed by bright field imageanalysis using white light (WL) or by fluorescence using complementaryDNA oligonucleotides attached to fluorophore-based dyes. DNA templatescan be sequenced, e.g., using ligation-based sequencing.

FIG. 33A shows a schematic diagram of the slide with beads attachedthereto. Note that only a small proportion of the DNA template moleculesare attached to the slide. One micron beads (Dynabeads MyOneStreptavidin beads; Dynal Biotech, Inc., Product No. 650.01) were used.However, a wide variety of beads could be used.

FIG. 33B shows a population of beads attached to a slide. The lowerpanels show the same region of the slide under white light (left) andfluorescence microscopy. The upper panel shows a range of beaddensities.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. In the following claimsarticles such as “a,”, “an” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims is introduced into another claim. Inparticular, any claim that is dependent on another claim can be modifiedto include one or more limitations found in any other claim that isdependent on the same base claim.

In addition, it is to be understood that any one or more embodiments maybe explicitly excluded from the claims even if the specific exclusion isnot set forth explicitly herein. It should also be understood that wherethe specification and/or claims disclose a reagent (e.g., a template,microsphere, probe, probe family, etc.) of use in sequencing, suchdisclosure also encompasses methods for sequencing using the reagentaccording either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise, or unless otherwise indicated in thespecification. In addition, where the specification and/or claimsdisclose a method of sequencing, any one or more of the reagentsdisclosed herein may be used in the method, unless one of ordinary skillin the art would understand otherwise, or unless use of the reagent insuch method is explicitly excluded in the specification. It shouldfurther be understood that where particular components of use insequencing are disclosed in the specification or claims, the inventionencompasses methods for making the reagents also. The term “component”is used broadly to refer to any item used in sequencing, includingtemplates, microparticles having templates attached thereto, libraries,etc. Furthermore, the figures are an integral part of the specification,and the invention includes structures shown in the figures, e.g.,microparticles having templates attached thereto, and methods disclosedin the figures.

Where ranges are given herein, the endpoints are included. Furthermore,it is to be understood that unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in different embodiments ofthe invention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

We claim:
 1. A method for determining information about a templatesequence, the method comprising the steps of: (I) performing asequencing reaction on a probe-template complex having a duplex portion,wherein the sequencing reaction comprises: (a) contacting the complexwith a collection of oligonucleotide probes, the collection comprisingat least two probe families, wherein: (i) a probe family is a group ofone or more different probes that are identically labeled, and (ii) theprobe families are labeled distinguishably from each other; (b)extending the duplex portion by ligating an extendable terminus of theduplex portion with a labeled probe from the collection that hashybridized to the template; (c) detecting the label of the ligated probeto identify its probe family , wherein the identity of the probe familyeliminates one or more possibilities for the identity of at least onetemplate sequence nucleotide but leaves at least two such possibilitiesopen for that nucleotide; and (d) repeating steps (a) to (c) for adesired number of cycles to obtain an ordered series of probe familynames of successively ligated probes, (II) optionally performing atleast one more sequencing reaction; and (III) generating an ordered listof probe family names from at least one ordered series of probe familynames from a sequencing reaction, wherein the ordered list providesinformation about the template sequence.
 2. The method of claim 1,wherein the method comprises using the ordered list from step (III) todetermine the identity of one or more nucleotides in the templatesequence.
 3. The method of claim 1, wherein the probe-template complexis initially formed by a process comprising: (a) rendering the templatesequence single-stranded if in double-stranded form, and (b) hybridizingan initializing oligonucleotide to a single-stranded portion of thetemplate sequence; and wherein the ordered list of probe family namesfrom step (III) is assembled by combining multiple ordered series ofprobe family names obtained from a plurality of sequencing reactionsusing multiple initializing oligonucleotides that initiate fromdifferent sites in the template.
 4. The method of claim 1, wherein theoligonucleotide probes in one or more probe families comprise anon-extendable moiety at one terminus.
 5. The method of claim 1, furthercomprising producing a new extendable terminus on the oligonucleotideprobe ligated in step (b) such that the new terminus generated isdifferent from the original terminus of the ligated probe beforeligation.
 6. The method of claim 1, wherein at least one oligonucleotideprobe comprises a phosphorothiolate linkage and the extendable terminusof the duplex is produced by cleaving the phosphorothiolate linkage witha cleavage agent comprising an atom selected from the group consistingof: Ag, Hg, Cu, Mn, Zn and Cd.
 7. The method of claim 1, wherein thecollection comprises 2distinguishably labeled probe families.
 8. Themethod of claim 1, wherein the collection comprises 3distinguishablylabeled probe families.
 9. The method of claim 1, wherein the collectioncomprises 4distinguishably labeled probe families.
 10. The method ofclaim 1, wherein the collection comprises more than 4 distinguishablylabeled probe families.
 11. The method of claim 1, wherein at least oneprobe in a probe family comprises a constrained portion in whichnucleotides are not selected independently of each other, in such amanner that knowing the identity of one or more nucleotides in theconstrained portion of the probe provides sufficient information toeliminate one or more possible identities for a nucleotide at one of theother positions.
 12. The method of claim 1, wherein probes are assignedto four probe families according to a scheme set forth in Table
 1. 13.The method of claim 11, wherein at least one nucleotide in the templatehas a known identity, and wherein the method comprises: (i) assigning atleast one identity to an unknown nucleotide in the template adjacent tothe nucleotide of known identity by determining which identity for theunknown nucleotide is consistent with (i) the identity of the knownnucleotide and (ii) the possible sequences of the constrained portion ofa probe whose proximal nucleotide is ligated opposite the unknownnucleotide on the template adjacent to the nucleotide of known identity;(ii) assigning at least one identity to a succeeding nucleotide in thetemplate by determining which identity is consistent with possiblesequences of the constrained portion of the probe whose proximalnucleotide is ligated opposite the succeeding nucleotide; and (iii)repeating step (ii) until the sequence information is determined. 14.The method of claim 1, further comprising the step of capping unligatedextendable termini on the duplex after ligation.
 15. The method of claim12, wherein the determining step comprises contacting the duplex with alabeled nucleotide in the presence of a polymerase under conditions thatallow incorporation of the labeled nucleotide if it is complementary tothe template at the position adjacent to the duplex.
 16. The method ofclaim 1 wherein the ordered list is generated from one or moresequencing reactions and method comprises: generating at least onepartial or complete candidate sequence from ordered list of probe familynames; and selecting a candidate sequence as the sequence of nucleotidesin the template.
 17. The method of claim 16, wherein the generating stepcomprises generating at least 4 candidate sequences.
 18. The method ofclaim 1, wherein the method comprises: (i) assuming an identity for afirst nucleotide in the sequence of nucleotides; (ii) assigning anidentity for a nucleotide adjacent to the first nucleotide bydetermining a possible identity for the adjacent nucleotide based on theprobe family information for the first nucleotide; (iii) assigning anidentity to a succeeding nucleotide by determining a possible identityfor the succeeding nucleotide based on the probe family information forthe nucleotide whose identity was most recently assigned; (iv) repeatingstep (iii) until a candidate sequence is generated; and (v) repeatingsteps (i)-(iv), wherein, in each repetition, a different identity isassumed for the first nucleotide, until a desired number of candidatesequences is generated.
 19. The method of claim 16, wherein theselecting step comprises comparing at least one candidate sequence withone or more known sequences and selecting a candidate sequence thatexhibits a predetermined degree of identity or is most nearly identicalto one or more of the known sequences.
 20. The method of claim 19,wherein the template is derived from an organism of interest, andwherein the comparing step comprises comparing at least one candidatesequence with sequences in a database that contains sequences obtainedfrom the organism.